Nucleic acids encoding CR2 polypeptides, vector and transformed cell thereof, and expression thereof

ABSTRACT

Early-induced genes by interleukin-2 (IL-2) have various DNA sequences. This patent describes a polyribonucleotide with a nucleotide segment encoding amino acids 1-60 of SEQ. ID No: 4, antibody binding homologues thereof, antibody binding fragments thereof at least 5 amino acids long, and fusion proteins thereof, alleles or naturally occurring mutants of the polyribonucleotide, and anti-sense polyribonucleotides thereof. Also provided are proteins, homologues, fragments, fusion proteins, vectors, transfected hosts, animal models, probes, and other related technology.

GOVERNMENT SUPPORT

Work described herein was supported in part by funding from the NationalInstitute of Health, Grant number 5 RO1-AI-32031-20. The United StatesGovernment has certain rights in the invention.

RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 08/330,108, filed Oct. 27, 1994 (now abandoned),which is a continuation application of U.S. patent application Ser. No.08/104,736, filed Aug. 10, 1993 (now abandoned), which is in turn acontinuation application of U.S. patent application Ser. No. 07/796,066,filed Nov. 20, 1991 (now abandoned). The contents of these priorapplications are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Mammalian cell growth, differentiation, and migration are directed byhormones and specific protein ligands, often termed cytokines. Inparticular, cells comprising the neuro-endocrine, hematopoietic and theimmune/inflammatory systems are known to be governed by cytokines.Cytokines, like other ligands, interact with cells by means of specificreceptors, usually expressed on the cell surface.

A fundamental problem confronting biomedical scientists is to discernhow signals are transduced through ligand receptors and how thesesignals determine the response of the cell. Many ligands influence theirtarget cells by stimulating the expression of specific genes. However,the genes signaled by most cytokines remain largely unknown owing to thecomplexity of cellular biochemistry. Moreover, the gene products thatare vital for performing different cellular processes are often onlyexpressed transiently, and/or in very low concentrations so that theyare difficult to detect, isolate and characterize.

Interleukin-2 (IL-2) is a cytokine that is critical for the immunesystem: it directs the proliferation and differentiation of Tlymphocytes (T-cells), B lymphocytes (B-cells), and natural killer (NK)cells. Just how IL-2 signals these cellular events in the various typesof target cells remains unknown. A few genes have been identified thatare expressed as a result of IL-2 stimulation of T cells. These includethe cellular proto-oncogenes c-fos, c-myb, c-myc, pim-1, and c-raf-1.However, exactly how many and what other genes are expressed as a resultof IL-2/IL-2 receptor interaction remains unknown.

Since the discovery of DNA cloning, methods have become available toisolate specific genes expressed by cells. However, it has beendifficult to devise new methods to isolate and clone all or most of thegenes expressed by a cell activated by a given ligand, a task that mustbe done before one can understand how the ligand directs the cell toperform specific functions. In addition, methods of identifying aparticular gene or genes stimulated early on after ligand receptoractivation have not been easily forthcoming as the number of genesstimulated by receptor activation from which a particular gene must beselected is usually quite large.

Therefore, what is needed are methods to select and enrich only forthose genes stimulated by a given ligand. Ideally, these methods shoulddetect those genes expressed in low concentrations, as well as thoseexpressed at high concentrations.

SUMMARY OF THE INVENTION

This invention pertains to complementary deoxyribonucleic acid (cDNA)libraries enriched in clones containing genes induced by ligandstimulation of a cell having a corresponding receptor for the ligand,and to methods of producing the same. This invention also relates to thegenes which are expressed immediately or early on as a consequence ofsuch a ligand-receptor interaction, and to methods of identifying thesegenes.

In the method of producing a cDNA library enriched in ligand-induciblegenes, a cellular ligand receptor on a cell is activated with a ligand,for a predetermined period of time, to induce expression of those genesexpressed as a result of ligand-receptor binding. Useful ligands includeany of those which can activate a specific cellular receptor. Theseinclude natural or synthetic ligands for the receptor. Ligands includecytokines such as the interleukins, cellular growth factors, colonystimulating factors, hormones, peptides, antibodies, andreceptor-binding fragments thereof.

The cells are activated with the ligand in the presence of labelled RNAprecursors. These precursors are incorporated into RNA synthesized bythe cell in response to receptor activation. Labelled precursors areused in order to distinguish newly transcribed RNA from unlabelled,preexisting RNA. Preferred labelled RNA precursors include6-thioguanine, 4-thiouridine, and tritiated uridine.

Activation is also carried out in the presence of a substance whichenhances the level of RNA in a cell. Preferred substances include theprotein synthesis inhibitors, cycloheximide and puromycin. Other usefulsubstances include cyclic 3',5'-adenosine monophosphate (cAMP), analogsof cAMP such as dibutyryl cAMP, and other molecules which increase theintracellular level of cAMP. The labelled RNA is then separated from theunlabelled RNA and used to prepare cDNA. The cDNA is cloned into hostcells to provide a cDNA library of cDNA-containing clones. This libraryis then screened for clones containing ligand-inducible genes.

In one embodiment of the invention, the screening step includes probingthe cDNA library with a DNA probe constructed from total cellular RNA ormRNA derived from (1) a ligand-induced cell and from (2) an uninducedcell. The library is probed under conditions such that the probehybridizes specifically with a complementary cDNA sequence in thelibrary. The selecting step includes selecting those clones containingsequences that hybridize only with probes constructed fromligand-induced mRNA or total RNA.

By following the method of the invention, eight clones containingligand-induced genes have been identified. These genes have been namedCytokine Response (CR) genes 1-8. CR genes 1-3, 5, 6, and 8 are novel.CR4 is identical to a gene reported as SATB-1 (Dickinson, L. A. et al.(1992) Cell 70:631-645), for Special AT-rich Binding protein 1, whichbinds selectively to the nuclear matrix/scaffold-associating region ofDNA. CR7, also identified using the method of the invention, isidentical to the putative proto-oncogene, pim 1, a known IL-2-inducedgene. The nucleic acid sequences of these CR genes, i.e., CR genes 1-6and 8 are set forth in SEQ ID NOs:1, 3, 5, 7, 9, 11, and 13 and FIGS.1-7. The amino acid sequences encoded by these CR genes are set forth inSEQ ID NOs.:2, 4, 6, 8, 10, 12, and 14 as well as in SEQ ID NOs:1, 3, 5,7, 9, 11, and 13 and FIGS. 1-7.

The present invention, therefore, also pertains to a CR1 polypeptide,preferably a substantially pure preparation of a CR1 polypeptide, or arecombinant CR1 polypeptide. In preferred embodiments, the CR1polypeptide has an amino acid sequence at least 60%, 80%, 90% or 95%homologous to the amino acid sequence in SEQ ID NO:2; the polypeptidehas an amino acid sequence essentially the same as the amino acidsequence in SEQ ID NO:2; the polypeptide is at least 5, 10, 20, 50, 100,or 150 amino acids in length; the polypeptide comprises at least 5,preferably at least 10, more preferably at least 20, more preferably atleast 50, 100, or 150 contiguous amino acids from SEQ ID NO:2. Infurther preferred embodiments, a protein homologous to SEQ ID NO:2 has amolecular weight of about 22 kilodaltons (kD), e.g. in the range of15-30 kD.

In a preferred embodiment, a polypeptide having at least one biologicalactivity of the CR1 polypeptide may differ in amino acid sequence fromthe sequence in SEQ ID NO:2, but such differences result in a modifiedpolypeptide which functions in the same or similar manner as native CR1protein or which has the same or similar characteristics of the nativeCR1 protein. Such a peptide can include at least 1, 2, 3, or 5, andpreferably 10, 20, and 30, amino acid residues from residues 1-202 ofSEQ ID NO:2.

In yet other preferred embodiments, the CR1 polypeptide is a recombinantfusion protein which includes a second polypeptide portion, e.g., asecond polypeptide having an amino acid sequence unrelated to a proteinrepresented by SEQ ID NO:2, e.g., the second polypeptide portion isglutathione-S-transferase, e.g., the second polypeptide portion is a DNAbinding domain, e.g., the second polypeptide portion is a polymeraseactivating domain, e.g. the fusion protein is functional in a two-hybridassay.

Yet another aspect of the present invention concerns an immunogencomprising a CR1 polypeptide in an immunogenic preparation, theimmunogen being capable of eliciting an immune response specific for theCR1 polypeptide; e.g., a humoral response, e.g. an antibody response;e.g. a cellular response. In preferred embodiments, the immunogencomprises an antigenic determinant, e.g., a unique determinant, from aprotein represented by SEQ ID NO:2. A further aspect of the presentinvention features an antibody preparation specifically reactive with anepitope of the CR1 immunogen.

Another aspect of the present invention provides a substantially purenucleic acid having a nucleotide sequence which encodes a CR1polypeptide. In preferred embodiments: the encoded polypeptide has atleast one biological activity; the encoded polypeptide has an amino acidsequence at least 60%, 80%, 90% or 95% homologous to the amino acidsequence in SEQ ID NO:2; the encoded polypeptide has an amino acidsequence essentially the same as the amino acid sequence in SEQ ID NO:2;the encoded polypeptide is at least 5, 10, 20, 50, 100, or 150 aminoacids in length; the encoded polypeptide comprises at least 5,preferably at least 10, more preferably at least 20, more preferably atleast 50, 100, or 150 contiguous amino acids from SEQ ID NO:2.

In a preferred embodiment, the encoded polypeptide having at least onebiological activity of the CR1 polypeptide may differ in amino acidsequence from the sequence in SEQ ID NO:2, but such differences resultin a modified polypeptide which functions in the same or similar manneras the native CR1 or which has the same or similar characteristics ofthe native CR1.

In yet other preferred embodiments, the encoded CR1 polypeptide is arecombinant fusion protein which includes a second polypeptide portion,e.g., a second polypeptide having an amino acid sequence unrelated to aprotein represented by SEQ ID NO:2, e.g., the second polypeptide portionis glutathione-S-transferase, e.g. the second polypeptide portion is aDNA binding domain, e.g., the second polypeptide portion is a polymeraseactivating domain, e.g., the fusion protein is functional in atwo-hybrid assay.

Furthermore, in certain preferred embodiments, the subject CR1 nucleicacid includes a transcriptional regulatory sequence, e.g. at least oneof a transcriptional promoter or transcriptional enhancer sequence,operably linked to the CR1 gene sequence, e.g., to render the CR1 genesequence suitable for use as an expression vector.

In yet a further preferred embodiment, the nucleic acid which encodes aCR1 polypeptide of the invention hybridizes under stringent conditionsto a nucleic acid probe corresponding to at least 12 consecutivenucleotides of SEQ ID NO:1; more preferably to at least 20 consecutivenucleotides of SEQ ID NO:1; more preferably to at least 40 consecutivenucleotides of SEQ ID NO:1. In yet a further preferred embodiment, theCR1 encoding nucleic acid hybridizes to a nucleic acid probecorresponding to a subsequence encoding at least 4 consecutive aminoacids, more preferably at least 10 consecutive amino acid residues, andeven more preferably at least 20 amino acid residues between residues1-202 of SEQ ID NO:2.

In preferred embodiments: the nucleic acid sequence includes at least 1,2, 3 or 5, and preferably at least 10, 20, 50, or 100 nucleotides fromthe region of SEQ ID NO:1 which encodes amino acid residues 1-202 of SEQID NO:2; the encoded peptide includes at least 1, 2, 3, 5, 10, 20, or 30amino acid residues from amino acid residues 1-202 of SEQ ID NO:2.

The present invention also pertains to a CR2 polypeptide, preferably asubstantially pure preparation of a CR2 polypeptide, or a recombinantCR2 polypeptide. In preferred embodiments, the CR2 polypeptide has anamino acid sequence at least 60%, 80%, 90% or 95% homologous to theamino acid sequence in SEQ ID NO:4; the polypeptide has an amino acidsequence essentially the same as the amino acid sequence in SEQ ID NO:4;the polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acids inlength; the polypeptide comprises at least 5, preferably at least 10,more preferably at least 20, more preferably at least 50, 100, or 150contiguous amino acids from SEQ ID NO:4. In further preferredembodiments, a protein homologous to SEQ ID NO:4 has a molecular weightof about 6 kilodaltons (kD), e.g. in the range of 5-15 kD.

In a preferred embodiment, a polypeptide having at least one biologicalactivity of the CR2 polypeptide may differ in amino acid sequence fromthe sequence in SEQ ID NO:4, but such differences result in a modifiedpolypeptide which functions in the same or similar manner as native CR2protein or which has the same or similar characteristics of the nativeCR2 protein. Such a peptide can include at least 1, 2, 3, or 5, andpreferably 10, 20, and 30, amino acid residues from residues 1-60 of SEQID NO:4.

In yet other preferred embodiments, the CR2 polypeptide is a recombinantfusion protein which includes a second polypeptide portion, e.g., asecond polypeptide having an amino acid sequence unrelated to a proteinrepresented by SEQ ID NO:4, e.g., the second polypeptide portion isglutathione-S-transferase, e.g., the second polypeptide portion is a DNAbinding domain, e.g., the second polypeptide portion is a polymeraseactivating domain, e.g., the fusion protein is functional in atwo-hybrid assay.

Yet another aspect of the present invention concerns an immunogencomprising a CR2 polypeptide in an immunogenic preparation, theimmunogen being capable of eliciting an immune response specific for theCR2 polypeptide; e.g. a humoral response, e.g. an antibody response;e.g. a cellular response. In preferred embodiments, the immunogencomprises an antigenic determinant, e.g. a unique determinant, from aprotein represented by SEQ ID NO:4. A further aspect of the presentinvention features an antibody preparation specifically reactive with anepitope of the CR2 immunogen.

Another aspect of the present invention provides a substantially purenucleic acid having a nucleotide sequence which encodes a CR2polypeptide. In preferred embodiments: the encoded polypeptide has atleast one biological activity; the encoded polypeptide has an amino acidsequence at least 60%, 80%, 90% or 95% homologous to the amino acidsequence in SEQ ID NO:4; the encoded polypeptide has an amino acidsequence essentially the same as the amino acid sequence in SEQ ID NO:4;the encoded polypeptide is at least 5, 10, 20, 50, 100, or 150 aminoacids in length; the encoded polypeptide comprises at least 5,preferably at least 10, more preferably at least 20, more preferably atleast 50, 100, or 150 contiguous amino acids from SEQ ID NO:4.

In a preferred embodiment, the encoded polypeptide having at least onebiological activity of the CR2 polypeptide may differ in amino acidsequence from the sequence in SEQ ID NO:4, but such differences resultin a modified polypeptide which functions in the same or similar manneras the native CR2 protein or which has the same or similarcharacteristics of the native CR2 protein.

In yet other preferred embodiments, the encoded CR2 polypeptide is arecombinant fusion protein which includes a second polypeptide portion,e.g., a second polypeptide having an amino acid sequence unrelated to aprotein represented by SEQ ID NO:4, e.g. the second polypeptide portionis glutathione-S-transferase, e.g., the second polypeptide portion is aDNA binding domain, e.g., the second polypeptide portion is a polymeraseactivating domain, e.g., the fusion protein is functional in atwo-hybrid assay.

Furthermore, in certain preferred embodiments, the subject CR2 nucleicacid includes a transcriptional regulatory sequence, e.g. at least oneof a transcriptional promoter or transcriptional enhancer sequence,operably linked to the CR2 gene sequence, e.g., to render the CR2 genesequence suitable for use as an expression vector.

In yet a further preferred embodiment, the nucleic acid which encodes aCR2 polypeptide of the invention hybridizes under stringent conditionsto a nucleic acid probe corresponding to at least 12 consecutivenucleotides of SEQ ID NO:3; more preferably to at least 20 consecutivenucleotides of SEQ ID NO:3; more preferably to at least 40 consecutivenucleotides of SEQ ID NO:3. In yet a further preferred embodiment, theCR2 encoding nucleic acid hybridizes to a nucleic acid probecorresponding to a subsequence encoding at least 4 consecutive aminoacids, more preferably at least 10 consecutive amino acid residues, andeven more preferably at least 20 amino acid residues between residues1-60 of SEQ ID NO:4.

In preferred embodiments: the nucleic acid sequence includes at least 1,2, 3 or 5, and preferably at least 10, 20, 50, or 100 nucleotides fromthe region of SEQ ID NO:3 which encodes amino acid residues 1-60 of SEQID NO:4; the encoded peptide includes at least 1, 2, 3, 5, 10, 20, or 30amino acid residues from amino acid residues 1-60 of SEQ ID NO:4.

The present invention further pertains to a CR3 polypeptide, preferablya substantially pure preparation of a CR3 polypeptide, or a recombinantCR3 polypeptide. In preferred embodiments, the CR3 polypeptide has anamino acid sequence at least 60%, 80%, 90% or 95% homologous to theamino acid sequence in SEQ ID NO:6; the polypeptide has an amino acidsequence essentially the same as the amino acid sequence in SEQ ID NO:6;the polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acids inlength; the polypeptide comprises at least 5, preferably at least 10,more preferably at least 20, more preferably at least 50, 100, or 150contiguous amino acids from SEQ ID NO:6. In further preferredembodiments, a protein homologous to SEQ ID NO:6 has a molecular weightof about 88 kilodaltons (kD), e.g. in the range of 80-95 kD.

In a preferred embodiment, a peptide having at least one biologicalactivity of the CR3 polypeptide may differ in amino acid sequence fromthe sequence in SEQ ID NO:6, but such differences result in a modifiedprotein which functions in the same or similar manner as native CR3protein or which has the same or similar characteristics of the nativeCR3 protein. Such a peptide can include at least 1, 2, 3, or 5, andpreferably 10, 20, and 30, amino acid residues from residues 1-358 ofSEQ ID NO:6.

In yet other preferred embodiments, the CR3 polypeptide is a recombinantfusion protein which includes a second polypeptide portion, e.g., asecond polypeptide having an amino acid sequence unrelated to a proteinrepresented by SEQ ID NO:6, e.g., the second polypeptide portion isglutathione-S-transferase, e.g., the second polypeptide portion is a DNAbinding domain, e.g., the second polypeptide portion is a polymeraseactivating domain, e.g., the fusion protein is functional in atwo-hybrid assay.

Yet another aspect of the present invention concerns an immunogencomprising a CR3 polypeptide in an immunogenic preparation, theimmunogen being capable of eliciting an immune response specific forsaid CR3 polypeptide; e.g. a humoral response, e.g. an antibodyresponse; e.g. a cellular response. In preferred embodiments, theimmunogen comprises an antigenic determinant, e.g. a unique determinant,from a protein represented by SEQ ID NO:6. A further aspect of thepresent invention features an antibody preparation specifically reactivewith an epitope of the CR3 immunogen.

Another aspect of the present invention provides a substantially purenucleic acid having a nucleotide sequence which encodes a CR3polypeptide. In preferred embodiments: the encoded polypeptide has atleast one biological activity; the encoded polypeptide has an amino acidsequence at least 60%, 80%, 90% or 95% homologous to the amino acidsequence in SEQ ID NO:6; the encoded polypeptide has an amino acidsequence essentially the same as the amino acid sequence in SEQ ID NO:6;the encoded polypeptide is at least 5, 10, 20, 50, 100, or 150 aminoacids in length; the encoded polypeptide comprises at least 5,preferably at least 10, more preferably at least 20, more preferably atleast 50, 100, or 150 contiguous amino acids from SEQ ID NO:6.

In a preferred embodiment, the encoded polypeptide having at least onebiological activity of the CR3 polypeptide may differ in amino acidsequence from the sequence in SEQ ID NO:6, but such differences resultin a modified polypeptide which functions in the same or similar manneras the native CR3 protein or which has the same or similarcharacteristics of the native CR3 protein.

In yet other preferred embodiments, the encoded CR3 polypeptide is arecombinant fusion protein which includes a second polypeptide portion,e.g., a second polypeptide having an amino acid sequence unrelated to aprotein represented by SEQ ID NO:6, e.g., the second polypeptide portionis glutathione-S-transferase, e.g., the second polypeptide portion is aDNA binding domain, e.g., the second polypeptide portion is a polymeraseactivating domain, e.g., the fusion protein is functional in atwo-hybrid assay.

Furthermore, in certain preferred embodiments, the subject CR3 nucleicacid includes a transcriptional regulatory sequence, e.g. at least oneof a transcriptional promoter or transcriptional enhancer sequence,operably linked to the CR3 gene sequence, e.g., to render the CR3 genesequence suitable for use as an expression vector.

In yet a further preferred embodiment, the nucleic acid which encodes aCR3 polypeptide of the invention hybridizes under stringent conditionsto a nucleic acid probe corresponding to at least 12 consecutivenucleotides of SEQ ID NO:5; more preferably to at least 20 consecutivenucleotides of SEQ ID NO:5; more preferably to at least 40 consecutivenucleotides of SEQ ID NO:5. In yet a further preferred embodiment, theCR3 encoding nucleic acid hybridizes to a nucleic acid probecorresponding to a subsequence encoding at least 4 consecutive aminoacids, more preferably at least 10 consecutive amino acid residues, andeven more preferably at least 20 amino acid residues between residues1-358 of SEQ ID NO:6.

In preferred embodiments: the nucleic acid sequence includes at least 1,2, 3 or 5, and preferably at least 10, 20, 50, or 100 nucleotides fromthe region of SEQ ID NO:5 which encodes amino acid residues 1-358 of SEQID NO:6; the encoded peptide includes at least 1, 2, 3, 5, 10, 20, or 30amino acid residues from amino acid residues 1-358 of SEQ ID NO:6.

The present invention still further pertains to a CR4 polypeptide,preferably a substantially pure preparation of a CR4 polypeptide, or arecombinant CR4 polypeptide. In preferred embodiments, the CR4polypeptide has an amino acid sequence at least 60%, 80%, 90% or 95%homologous to the amino acid sequence in SEQ ID NO:8; the polypeptidehas an amino acid sequence essentially the same as the amino acidsequence in SEQ ID NO:8; the polypeptide is at least 5, 10, 20, 50, 100,or 150 amino acids in length; the polypeptide comprises at least 5,preferably at least 10, more preferably at least 20, more preferably atleast 50, 100, or 150 contiguous amino acids from SEQ ID NO:8. Infurther preferred embodiments, a protein homologous to SEQ ID NO:8 has amolecular weight of about 83 kilodaltons (kD), e.g. in the range of75-90 kD.

In a preferred embodiment, a polypeptide having at least one biologicalactivity of the CR4 polypeptide may differ in amino acid sequence fromthe sequence in SEQ ID NO:8, but such differences result in a modifiedpolypeptide which functions in the same or similar manner as native CR4protein or which has the same or similar characteristics of the nativeCR4 protein. Such a peptide can include at least 1, 2, 3, or 5, andpreferably 10, 20, and 30, amino acid residues from residues 1-763 ofSEQ ID NO:8.

In yet other preferred embodiments, the CR4 polypeptide is a recombinantfusion protein which includes a second polypeptide portion, e.g., asecond polypeptide having an amino acid sequence unrelated to a proteinrepresented by SEQ ID NO:8, e.g., the second polypeptide portion isglutathione-S-transferase, e.g., the second polypeptide portion is a DNAbinding domain, e.g., the second polypeptide portion is a polymeraseactivating domain, e.g., the fusion protein is functional in atwo-hybrid assay.

Yet another aspect of the present invention pertains to an immunogencomprising a CR4 polypeptide in an immunogenic preparation, theimmunogen being capable of eliciting an immune response specific for theCR4 polypeptide; e.g. a humoral response, e.g. an antibody response;e.g. a cellular response. In preferred embodiments, the immunogencomprises an antigenic determinant, e.g. a unique determinant, from aprotein represented by SEQ ID NO:8. A further aspect of the presentinvention features an antibody preparation specifically reactive with anepitope of the CR4 immunogen.

Another aspect of the present invention provides a substantially purenucleic acid having a nucleotide sequence which encodes a CR4polypeptide. In preferred embodiments: the encoded polypeptide has atleast one biological activity; the encoded polypeptide has an amino acidsequence at least 60%, 80%, 90% or 95% homologous to the amino acidsequence in SEQ ID NO:8; the encoded polypeptide has an amino acidsequence essentially the same as the amino acid sequence in SEQ ID NO:8;the encoded polypeptide is at least 5, 10, 20, 50, 100, or 150 aminoacids in length; the encoded polypeptide comprises at least 5,preferably at least 10, more preferably at least 20, more preferably atleast 50, 100, or 150 contiguous amino acids from SEQ ID NO:8.

In a preferred embodiment, the encoded polypeptide having at least onebiological activity of the CR4 polypeptide may differ in amino acidsequence from the sequence in SEQ ID NO:8, but such differences resultin a modified polypeptide which functions in the same or similar manneras the native CR4 protein or which has the same or similarcharacteristics of the native CR4 protein.

In yet other preferred embodiments, the encoded CR4 polypeptide is arecombinant fusion protein which includes a second polypeptide portion,e.g., a second polypeptide having an amino acid sequence unrelated to aprotein represented by SEQ ID NO:8, e.g., the second polypeptide portionis glutathione-S-transferase, e.g. the second polypeptide portion is aDNA binding domain, e.g., the second polypeptide portion is a polymeraseactivating domain, e.g., the fusion protein is functional in atwo-hybrid assay.

Furthermore, in certain preferred embodiments, the subject CR4 nucleicacid includes a transcriptional regulatory sequence, e.g., at least oneof a transcriptional promoter or transcriptional enhancer sequence,operably linked to the CR4 gene sequence, e.g., to render the CR4 genesequence suitable for use as an expression vector.

In yet a further preferred embodiment, the nucleic acid which encodes aCR4 polypeptide of the invention hybridizes under stringent conditionsto a nucleic acid probe corresponding to at least 12 consecutivenucleotides of SEQ ID NO:7; more preferably to at least 20 consecutivenucleotides of SEQ ID NO:7; more preferably to at least 40 consecutivenucleotides of SEQ ID NO:7. In yet a further preferred embodiment, theCR4 encoding nucleic acid hybridizes to a nucleic acid probecorresponding to a subsequence encoding at least 4 consecutive aminoacids, more preferably at least 10 consecutive amino acid residues, andeven more preferably at least 20 amino acid residues between residues1-763 of SEQ ID NO:8.

In preferred embodiments: the nucleic acid sequence includes at least 1,2, 3 or 5, and preferably at least 10, 20, 50, or 100 nucleotides fromthe region of SEQ ID NO:7 which encodes amino acid residues 1-763 of SEQID NO:8; the encoded peptide includes at least 1, 2, 3, 5, 10, 20, or 30amino acid residues from amino acid residues 1-763 of SEQ ID NO:8.

Another aspect of the present invention pertains to a CR5 polypeptide,preferably a substantially pure preparation of a CR5 polypeptide, or arecombinant CR5 polypeptide. In preferred embodiments, the CR5polypeptide has an amino acid sequence at least 60%, 80%, 90% or 95%homologous to the amino acid sequence in SEQ ID NO:10; the polypeptidehas an amino acid sequence essentially the same as the amino acidsequence in SEQ ID NO:10; the polypeptide is at least 5, 10, 20, 50,100, or 150 amino acids in length; the polypeptide comprises at least 5,preferably at least 10, more preferably at least 20, more preferably atleast 50, 100, or 150 contiguous amino acids from SEQ ID NO:10. Infurther preferred embodiments, a protein homologous to SEQ ID NO:10 hasa molecular weight of about 28 kilodaltons (kD), e.g. in the range of20-35 kD.

In a preferred embodiment, a polypeptide having at least one biologicalactivity of the CR5 polypeptide may differ in amino acid sequence fromthe sequence in SEQ ID NO:10, but such differences result in a modifiedpolypeptide which functions in the same or similar manner as native CR5protein or which has the same or similar characteristics of the nativeCR5 protein. Such a peptide can include at least 1, 2, 3, or 5, andpreferably 10, 20, and 30, amino acid residues from residues 1-258 ofSEQ ID NO:10.

In yet other preferred embodiments, the CR5 polypeptide is a recombinantfusion protein which includes a second polypeptide portion, e.g., asecond polypeptide having an amino acid sequence unrelated to a proteinrepresented by SEQ ID NO:10, e.g., the second polypeptide portion isglutathione-S-transferase, e.g. the second polypeptide portion is a DNAbinding domain, e.g., the second polypeptide portion is a polymeraseactivating domain, e.g., the fusion protein is functional in atwo-hybrid assay.

Yet another aspect of the present invention concerns an immunogencomprising a CR5 polypeptide in an immunogenic preparation, theimmunogen being capable of eliciting an immune response specific for theCR5 polypeptide; e.g. a humoral response, e.g. an antibody response;e.g. a cellular response. In preferred embodiments, the immunogencomprises an antigenic determinant, e.g. a unique determinant, from aprotein represented by SEQ ID NO:10. A further aspect of the presentinvention features an antibody preparation specifically reactive with anepitope of the CR5 immunogen.

Another aspect of the present invention provides a substantially purenucleic acid having a nucleotide sequence which encodes a CR5polypeptide. In preferred embodiments: the encoded polypeptide has atleast one biological activity; the encoded polypeptide has an amino acidsequence at least 60%, 80%, 90% or 95% homologous to the amino acidsequence in SEQ ID NO:10; the encoded polypeptide has an amino acidsequence essentially the same as the amino acid sequence in SEQ IDNO:10; the encoded polypeptide is at least 5, 10, 20, 50, 100, or 150amino acids in length; the encoded polypeptide comprises at least 5,preferably at least 10, more preferably at least 20, more preferably atleast 50, 100, or 150 contiguous amino acids from SEQ ID NO:10.

In a preferred embodiment, the encoded polypeptide having at least onebiological activity of the CR5 polypeptide may differ in amino acidsequence from the sequence in SEQ ID NO:10, but such differences resultin a modified polypeptide which functions in the same or similar manneras the native CR5 protein or which has the same or similarcharacteristics of the native CR5 protein.

In yet other preferred embodiments, the encoded CR5 polypeptide is arecombinant fusion protein which includes a second polypeptide portion,e.g., a second polypeptide having an amino acid sequence unrelated to aprotein represented by SEQ ID NO:10, e.g., the second polypeptideportion is glutathione-S-transferase, e.g., the second polypeptideportion is a DNA binding domain, e.g., the second polypeptide portion isa polymerase activating domain, e.g. the fusion protein is functional ina two-hybrid assay.

Furthermore, in certain preferred embodiments, the subject CR5 nucleicacid includes a transcriptional regulatory sequence, e.g., at least oneof a transcriptional promoter or transcriptional enhancer sequence,operably linked to the CR5 gene sequence, e.g., to render the CR5 genesequence suitable for use as an expression vector.

In yet a further preferred embodiment, the nucleic acid which encodes anCR5 polypeptide of the invention hybridizes under stringent conditionsto a nucleic acid probe corresponding to at least 12 consecutivenucleotides of SEQ ID NO:9; more preferably to at least 20 consecutivenucleotides of SEQ ID NO:9; more preferably to at least 40 consecutivenucleotides of SEQ ID NO:9. In yet a further preferred embodiment, theCR5 encoding nucleic acid hybridizes to a nucleic acid probecorresponding to a subsequence encoding at least 4 consecutive aminoacids, more preferably at least 10 consecutive amino acid residues, andeven more preferably at least 20 amino acid residues between residues1-258 of SEQ ID NO:10.

In preferred embodiments: the nucleic acid sequence includes at least 1,2, 3 or 5, and preferably at least 10, 20, 50, or 100 nucleotides fromthe region of SEQ ID NO:9 which encodes amino acid residues 1-258 of SEQID NO:10; the encoded peptide includes at least 1, 2, 3, 5, 10, 20, or30 amino acid residues from amino acid residues 1-258 of SEQ ID NO:10.

The present invention further pertains to a CR6 polypeptide, preferablya substantially pure preparation of a CR6 polypeptide, or a recombinantCR6 polypeptide. In preferred embodiments, the CR6 polypeptide has anamino acid sequence at least 60%, 80%, 90% or 95% homologous to theamino acid sequence in SEQ ID NO:12; the polypeptide has an amino acidsequence essentially the same as the amino acid sequence in SEQ IDNO:12; the polypeptide is at least 5, 10, 20, 50, 100, or 150 aminoacids in length; the polypeptide comprises at least 5, preferably atleast 10, more preferably at least 20, more preferably at least 50, 100,or 150 contiguous amino acids from SEQ ID NO:12. In further preferredembodiments, a protein homologous to SEQ ID NO:12 has a molecular weightof about 17 kilodaltons (kD), e.g. in the range of 15-25 kD.

In a preferred embodiment, a polypeptide having at least one biologicalactivity of the CR6 polypeptide may differ in amino acid sequence fromthe sequence in SEQ ID NO:12, but such differences result in a modifiedpolypeptide which functions in the same or similar manner as native CR6protein or which has the same or similar characteristics of the nativeCR6 protein. Such a peptide can include at least 1, 2, 3, or 5, andpreferably 10, 20, and 30, amino acid residues from residues 1-159 ofSEQ ID NO:12.

In yet other preferred embodiments, the CR6 polypeptide is a recombinantfusion protein which includes a second polypeptide portion, e.g., asecond polypeptide having an amino acid sequence unrelated to a proteinrepresented by SEQ ID NO:12, e.g., the second polypeptide portion isglutathione-S-transferase, e.g., the second polypeptide portion is a DNAbinding domain, e.g., the second polypeptide portion is a polymeraseactivating domain, e.g. the fusion protein is functional in a two-hybridassay.

Yet another aspect of the present invention concerns an immunogencomprising a CR6 polypeptide in an immunogenic preparation, theimmunogen being capable of eliciting an immune response specific for theCR6 polypeptide; e.g. a humoral response, e.g. an antibody response;e.g. a cellular response. In preferred embodiments, the immunogencomprises an antigenic determinant, e.g. a unique determinant, from aprotein represented by SEQ ID NO:12. A further aspect of the presentinvention features an antibody preparation specifically reactive with anepitope of the CR6 immunogen.

Another aspect of the present invention provides a substantially purenucleic acid having a nucleotide sequence which encodes a CR6polypeptide. In preferred embodiments: the encoded polypeptide has atleast one biological activity; the encoded polypeptide has an amino acidsequence at least 60%, 80%, 90% or 95% homologous to the amino acidsequence in SEQ ID NO:12; the encoded polypeptide has an amino acidsequence essentially the same as the amino acid sequence in SEQ IDNO:12; the encoded polypeptide is at least 5, 10, 20, 50, 100, or 150amino acids in length; the encoded polypeptide comprises at least 5,preferably at least 10, more preferably at least 20, more preferably atleast 50, 100, or 150 contiguous amino acids from SEQ ID NO:12.

In a preferred embodiment, the encoded polypeptide having at least onebiological activity of the CR6 polypeptide may differ in amino acidsequence from the sequence in SEQ ID NO:12, but such differences resultin a modified polypeptide which functions in the same or similar manneras the native CR6 protein or which has the same or similarcharacteristics of the native CR6 protein.

In yet other preferred embodiments, the encoded CR6 polypeptide is arecombinant fusion protein which includes a second polypeptide portion,e.g., a second polypeptide having an amino acid sequence unrelated to aprotein represented by SEQ ID NO:12, e.g., the second polypeptideportion is glutathione-S-transferase, e.g., the second polypeptideportion is a DNA binding domain, e.g., the second polypeptide portion isa polymerase activating domain, e.g., the fusion protein is functionalin a two-hybrid assay.

Furthermore, in certain preferred embodiments, the subject CR6 nucleicacid includes a transcriptional regulatory sequence, e.g. at least oneof a transcriptional promoter or transcriptional enhancer sequence,operably linked to the CR6 gene sequence, e.g., to render the CR6 genesequence suitable for use as an expression vector.

In yet a further preferred embodiment, the nucleic acid which encodes anCR6 polypeptide of the invention hybridizes under stringent conditionsto a nucleic acid probe corresponding to at least 12 consecutivenucleotides of SEQ ID NO:11; more preferably to at least 20 consecutivenucleotides of SEQ ID NO:11; more preferably to at least 40 consecutivenucleotides of SEQ ID NO:11. In yet a further preferred embodiment, theCR6 encoding nucleic acid hybridizes to a nucleic acid probecorresponding to a subsequence encoding at least 4 consecutive aminoacids, more preferably at least 10 consecutive amino acid residues, andeven more preferably at least 20 amino acid residues between residues1-159 of SEQ ID NO:12.

In preferred embodiments: the nucleic acid sequence includes at least 1,2, 3 or 5, and preferably at least 10, 20, 50, or 100 nucleotides fromthe region of SEQ ID NO:11 which encodes amino acid residues 1-159 ofSEQ ID NO:12; the encoded peptide includes at least 1, 2, 3, 5, 10, 20,or 30 amino acid residues from amino acid residues 1-159 of SEQ IDNO:12.

The present invention still further pertains to a CR8 polypeptide,preferably a substantially pure preparation of a CR8 polypeptide, or arecombinant CR8 polypeptide. In preferred embodiments, the CR8polypeptide has an amino acid sequence at least 60%, 80%, 90% or 95%homologous to the amino acid sequence in SEQ ID NO:14; the polypeptidehas an amino acid sequence essentially the same as the amino acidsequence in SEQ ID NO:14; the polypeptide is at least 5, 10, 20, 50,100, or 150 amino acids in length; the polypeptide comprises at least 5,preferably at least 10, more preferably at least 20, more preferably atleast 50, 100, or 150 contiguous amino acids from SEQ ID NO:14. Infurther preferred embodiments, a protein homologous to SEQ ID NO:14 hasa molecular weight of about 45 kilodaltons (kD), e.g. in the range of35-50 kD.

In a preferred embodiment, a polypeptide having at least one biologicalactivity of the CR8 polypeptide may differ in amino acid sequence fromthe sequence in SEQ ID NO:14, but such differences result in a modifiedpolypeptide which functions in the same or similar manner as native CR8protein or which has the same or similar characteristics of the nativeCR8 protein. Such a peptide can include at least 1, 2, 3, or 5, andpreferably 10, 20, and 30, amino acid residues from residues 1-412 ofSEQ ID NO:14.

In yet other preferred embodiments, the CR8 polypeptide is a recombinantfusion protein which includes a second polypeptide portion, e.g., asecond polypeptide having an amino acid sequence unrelated to a proteinrepresented by SEQ ID NO:14, e.g., the second polypeptide portion isglutathione-S-transferase, e.g., the second polypeptide portion is a DNAbinding domain, e.g., the second polypeptide portion is a polymeraseactivating domain, e.g., the fusion protein is functional in atwo-hybrid assay.

Yet another aspect of the present invention pertains to an immunogencomprising a CR8 polypeptide in an immunogenic preparation, theimmunogen being capable of eliciting an immune response specific forsaid CR8 polypeptide; e.g. a humoral response, e.g. an antibodyresponse; e.g. a cellular response. In preferred embodiments, theimmunogen comprises an antigenic determinant, e.g. a unique determinant,from a protein represented by SEQ ID NO:14. A further aspect of thepresent invention features an antibody preparation specifically reactivewith an epitope of the CR8 immunogen.

Another aspect of the present invention provides a substantially purenucleic acid having a nucleotide sequence which encodes a CR8polypeptide. In preferred embodiments: the encoded polypeptide has atleast one biological activity; the encoded polypeptide has an amino acidsequence at least 60%, 80%, 90% or 95% homologous to the amino acidsequence in SEQ ID NO:14; the encoded polypeptide has an amino acidsequence essentially the same as the amino acid sequence in SEQ IDNO:14; the encoded polypeptide is at least 5, 10, 20, 50, 100, or 150amino acids in length; the encoded polypeptide comprises at least 5,preferably at least 10, more preferably at least 20, more preferably atleast 50, 100, or 150 contiguous amino acids from SEQ ID NO:14.

In a preferred embodiment, the encoded polypeptide having at least onebiological activity of the CR8 polypeptide may differ in amino acidsequence from the sequence in SEQ ID NO:14, but such differences resultin a modified polypeptide which functions in the same or similar manneras the native CR8 protein or which has the same or similarcharacteristics of the native CR8 protein.

In yet other preferred embodiments, the encoded CR8 polypeptide is arecombinant fusion protein which includes a second polypeptide portion,e.g., a second polypeptide having an amino acid sequence unrelated to aprotein represented by SEQ ID NO:14, e.g., the second polypeptideportion is glutathione-S-transferase, e.g., the second polypeptideportion is a DNA binding domain, e.g., the second polypeptide portion isa polymerase activating domain, e.g. the fusion protein is functional ina two-hybrid assay.

Furthermore, in certain preferred embodiments, the subject CR8 nucleicacid includes a transcriptional regulatory sequence, e.g., at least oneof a transcriptional promoter or transcriptional enhancer sequence,operably linked to the CR8 gene sequence, e.g., to render the CR8 genesequence suitable for use as an expression vector.

In yet a further preferred embodiment, the nucleic acid which encodes aCR8 polypeptide of the invention hybridizes under stringent conditionsto a nucleic acid probe corresponding to at least 12 consecutivenucleotides of SEQ ID NO:13; more preferably to at least 20 consecutivenucleotides of SEQ ID NO:13; more preferably to at least 40 consecutivenucleotides of SEQ ID NO:13. In yet a further preferred embodiment, theCR8 encoding nucleic acid hybridizes to a nucleic acid probecorresponding to a subsequence encoding at least 4 consecutive aminoacids, more preferably at least 10 consecutive amino acid residues, andeven more preferably at least 20 amino acid residues between residues1-412 of SEQ ID NO:14.

In preferred embodiments: the nucleic acid sequence includes at least 1,2, 3 or 5, and preferably at least 10, 20, 50, or 100 nucleotides fromthe region of SEQ ID NO:13 which encodes amino acid residues 1-412 ofSEQ ID NO:14; the encoded peptide includes at least 1, 2, 3, 5, 10, 20,or 30 amino acid residues from amino acid residues 1-412 of SEQ IDNO:14.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a histogram showing the level of DNA synthesis (as theincorporation of ³ H!-thymidine in PBMN cells treated with CHX, OKT3 orOKT3 and CHX.

FIG. 2 shows CR8 expression in the following cytokine-dependent celllines: the IL-2-dependent human T cell line Kit 225; the IL-3-dependentmouse pro-B cell line Ba/F3; and the IL-2-dependent mouse T cell lineCTLL2.

DETAILED DESCRIPTION OF THE INVENTION

By combining several different procedures, a cDNA library can beconstructed which is enriched in clones containing genes whoseexpression is induced by activation of a cellular ligand-specificreceptor. This enriched library can facilitate identification andcharacterization of ligand-activated genes that are triggeredimmediately and/or early on after receptor activation (e.g., 2 to 4hours after the ligand binds to its receptor). Such genes may play arole in stimulating growth phase transitions and subsequent clonalexpansion of a particular cell type.

The method of the invention can be used to create cDNA libraries of thegenes induced by activation of a variety of different cellularreceptors. The receptors can be cytoplasmic, nuclear, or cell-surfacereceptors, and include receptors for cytokines, hormones, factors, andpeptides. For example, cytokines such as the interleukins (e.g., IL-1and IL-2), cellular growth factors (e.g., platelet-derived growth factor(PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF),insulin-like growth factor (IGF)), colony stimulating factors (e.g.,multiplication stimulating activity), and hormones (e.g., insulin,somatomedin C, and steroid hormones are useful as activators of certaincellular receptors.

The ligand used to activate the receptor can be the natural ligandrecognized by the receptor or a synthetic analog. Alternatively, anantibody specific for the receptor and capable of activating thereceptor can be used.

The receptor is activated by a ligand or other activation for apredetermined length of time and at a concentration necessary toactivate the receptor. This activation is carried out in the presence oflabelled RNA precursors which are incorporated into the RNA synthesizedby the cell in response to receptor activation. Thus, the RNAtranscribed is labelled so as to be distinguished from preexisting RNAwhich is not labelled.

Some labels (such as radiolabels) can be employed to monitor the newlysynthesized RNA. Useful radiolabelled RNA precursors for such purposesinclude ³ H!-uridine. Other labels may be used to separate newlytranscribed RNA from unlabelled RNA. For example, RNA synthesized fromthiol-labelled RNA precursors specifically adheres to phenylmercuryagarose (Woodford et al. (1988) Anal. Biochem. 1781:166-172). RNA newlysynthesized in response to receptor activation can be separated frompreexisting RNA in the cell; all RNA molecules expressed prior toligand-activation pass through the phenylmethyl mercury column, leavingonly the newly synthesized, thiol-(SH--) labelled RNA attached to theagarose via a covalent bond between the mercury and sulfur. Thethiol-labelled RNA molecules are then eluted from the column by reducingthe Hg-S bond with an excess of 2-mercaptoethanol.

To augment the expression of immediate/early ligand-activated geneswhich may be difficult to identify because of the large number ofdownstream genes turned on at a later time, a substance that enhancesthe level of RNA is added to the culture medium during the ligandstimulation (see, e.g., Cochran et al. (1983) Cell 33:939-947). Usefulsubstances include those compounds that stabilize RNA and/or that blocktranslation, thereby blocking feedback inhibition of these genes by alater gene product. Such activity may potentiate the magnitude of theRNA expressed as well as the duration of the life of the RNA. Examplesof such useful substances include cyclohexamide (CHX), which inhibitsprotein synthesis at the level of RNA-ribosome complexing and maystabilize polysomal RNA, and puromycin, which inhibits translation bycausing premature dissociation of the peptide-mRNA-ribosome complex.

cAMP is another useful substance which enhances the level of RNA.Increased levels of cAMP, or analogs or agents that elevate cAMP levels,such as forskolin, dibutyryl AMP, and isobutylmethyl xanthene, are knownto inhibit cell growth, proliferation, and inositol phospholipidturnover. In addition, elevated levels of cAMP completely inhibitIL-2-stimulated T-cell proliferation (Johnson et al. (1988) Proc. Natl.Acad. Sci. (USA) 85:6072-6076).

The labelled RNA transcribed consequent to receptor activation in thepresence of the substance which enhances RNA levels is then purifiedfrom the cytoplasm of the cells. Purification can be accomplished byextracting total cellular RNA from a cell homogenate or fractionthereof, isolating mRNA therefrom, for example, using a poly U or polydT! column, and then separating the labelled RNA from the unlabelledRNA. Separation can be accomplished, for example, using the phenylmethylmercury agarose protocol described above. Of course, other known methodsof separating the newly synthesized RNA from the preexisting can also beused.

The cDNA libraries can be prepared from the separated labelled RNA bystandard techniques. For example, the labelled RNA may be reversedtranscribed into cDNA, using oligo dT! primers. The cDNA is then ligatedinto appropriate vectors using established recombinant DNA techniques. AcDNA library is then constructed by methods well known in the art inprokaryotic or eukaryotic host cells that are capable of beingtransfected by the vectors.

Prokaryotic systems most commonly utilize E. coli as host, althoughother bacterial strains such as Bacillus, Pseudomonas, or otherGram-positive or Gram-negative prokaryotes can also be used. When suchprokaryotic hosts are employed, operable control systems compatible withthese hosts are ligated to the cDNA fragments and disposed on a suitabletransfer vector which is capable of replication in the bacterial hostcell. Backbone vectors capable of replication include phage vectors andplasmid vectors, as is known in the art. Common plasmid vectors includethose derived from pBR322 and the pUC series. One such useful vectorwhich is commercially available is the plasmid pBluescriptTIISK+(Stratagene, La Jolla, Calif.). Charon lambda phage is a frequentlyemployed phage vector. Control sequences obligatorily include promoterand ribosome binding site encoding sequences, and a variety of suchcontrols are available in the art, such as the beta-lactamase(pencillinase) and lactose (lac) promoter systems (see, e.g., Chang etal. (1977) Nature 198:106), and the tryptophan (trp) promoter systems(Goeddel et al. (1980) Nucleic Acids Res. 8:4057). Composite promoterscontaining elements of both the trp and lac promoter systems are alsoavailable in the art.

Eukaryotic microbes, such as laboratory strains of Saccharomycescerevisiae, or Baker's yeast, can also be used for expression. A numberof yeast control systems and vectors are available, including thosewhich are promoters for the synthesis of glycolytic enzymes (see, e.g.,Hess et al. (1968) Biochem. 17:4900). Yeast vectors employing the 2micron origin of replication are suitable as transfer vectors (see,e.g., Broach (1982) Meth. Enzym. 101:307).

Tissue cultures of insect cell lines, or cell lines immortalized frommammalian or other higher organisms have also been used as recombinanthosts. Such cell lines include chinese hamster ovary (CHO), Vero, HeLa,and COS cells. In general, the COS cell system is used for transientexpression, while CHO cells typically integrate transformed DNA into thechromosome. Suitable mammalian vectors are generally based on viralorigins of replication and control sequences. Most commonly used are thesimian virus 40 (SV40) promoters and replicons (see Fiers et al. (1978)Nature 273:113) and similar systems derived from Adenovirus 2, bovinepapilloma virus, and avian sarcoma virus.

The ligand-activated genes are then screened in the library using anyone of several different methods. One method involves differentialhybridization with cDNA probes constructed from mRNA derived fromligand-activated cells and unactivated cells. Another method includeshybridization subtraction, whereby cDNA from ligand-activated cells ishybridized with an excess of mRNA from unactivated cells to remove RNAmolecules common to both. Alternatively, cDNA probes can be made fromthe same pool of thiol-selected mRNA used to make the cDNA library, asthese sequences are highly enriched for ligand-induced molecules. Onecan prepare cDNA probes from mRNA extracted from cells treated withdrugs that block the biologic response to the particular cytokine (e.g.,rapamycin blocks the proliferative response of T cells to IL-2, andcyclosporin A and FK506 block the T-cell response to activation via theT-cell antigen receptor). Results from probing with the cDNA made fromdrug-inhibited cells can then be compared to results from probes madefrom cells not inhibited by these drugs.

The marked superinduction observed for a number of the genes using asubstance, such as CHX, which enhances RNA levels is crucial in enablingtheir detection by differential hybridization, as it has been estimatedthat differential hybridization is only effective in the detection ofrelatively high-abundance RNAs expressed at a level of greater than 500copies per cell. Therefore, the superinduction increases that level ofexpression of low-abundance RNAs above the threshold of detection bydifferential screening. In addition, the approximately 10-foldenrichment for newly synthesized RNA afforded by the thiol-labellingprocedure further heightens the efficacy of the cloning procedure. Thus,the combination of superinduction and thiol-labelling of RNAsignificantly enhances the sensitivity of differential screening, andprovides a cloning strategy which has the capacity to detect messagesnormally present in relatively low abundance (i.e., less than 100copies/cell).

After the initial screening of the cDNA library, all clones isolated astentatively positive must be corroborated as truly ligand-activated.This can be accomplished by isolating the cDNA insert from each clonedplasmid, and then employing this cDNA to probe RNA from ligand-activatedcells by Northern blot analysis.

Then, to identify each gene, the cDNA can be subjected to sequenceanalysis. Searches of the GenBank (Los Alamos, N.Mex.) and EMBL(Heidleberg, Germany) data bases can be made of even partial sequencesto identify known sequences such as pim-1, a previously characterized,IL-2 induced gene.

A number of methods can be used to characterize the novelligand-enhanced genes and begin to determine their functional roles in,for example, signal transduction. DNA sequence analysis of the cDNA ofthe mRNA transcript can predict the coding region for the gene productand the amino acid sequence. From the amino acid sequence, the geneproduct can be placed into one of several categories of proteins, suchas DNA-binding proteins, kinases, phosphatases, transmembrane proteins,or secreted products. These categories then will predict certain obviousfunctions and characteristics to be examined.

For example, the mechanism whereby IL-2 binding to its heterodimericp55/p75 receptor on the cell surface activates specific gene expressionis not well understood. The 75 kD component of the IL-2 receptor, whichis responsible for signal transduction, does not exhibit sequencehomologies indicative of previously characterized functional domains.However, the involvement of protein phosphorylation in the IL-2 responsehas been indicated by the activation of IL-2R-associated kinases,including the tyrosine kinase p56^(lck), as well as the cytoplasmicserine/threonine kinase c-raf-1 in early IL-2-mediated transmembranesignalling. In addition, a number of proteins, including the IL-2R p75,are rapidly phosphorylated in response to IL-2. The hydrolysis ofphosphatidylinositol glycan is also stimulated by IL-2, resulting in theformation of the putative second messengers myristylated diacylglyceroland inositol phosphate-glycan. Analysis of the regulatory elementsgoverning expression of the immediate-early genes described in thepresent study will be useful in the further characterization of thesecondary biochemical messengers activated by the IL-2 receptor.

Other methods helpful in determining the functional relevance of theIL-2-induced genes include examining T-cells for their expression inresponse to triggering of other receptors.

One such receptor is the T-cell antigen receptor. Seminal studies of theT-cell system have demonstrated that T-cell activation occurs as atwo-step process. Quiescent cells are initially stimulated throughengagement of the antigen receptor, which provides the cells with thecapacity to produce and respond to IL-2. Subsequently, the interactionof IL-2 with its cell-surface receptor drives progression through the G₁to the S phase of the cell cycle. Transmembrane signalling through boththe T-cell antigen receptor has been shown to trigger the heightenedexpression of a number of genes, including c-fos, c-myc and c-raf-1(Reed et al. (1986) Proc. Nat. Acad. Sci. USA 83:3982-3986; Dautry etal. (1988) J. Biol. Chem. 263:17615-17620; and Zmuidzinas et al. (1991)Mol. Cell. Biol. 11:2794-2803). By comparison, in the case of the c-mybgene, the induction is unique to the IL-2 signalling pathway (Stern etal. (1986) Science 233:203-206). Therefore, to categorize the novelIL-2-induced genes with regard to their patterns of induction by thesetwo receptor pathways, the sensitivity of the genes to T-cell receptorstimulation can be determined.

Additional methods that can be used to categorize the genes isolatedinclude screening for expression by proliferating versusnon-proliferating cells, for tissue-specific expression, and forexpression in response to different cytokines and hormones. Genes thatare expressed exclusively by proliferating cells are obvious candidatesfor functioning to promote cell growth. Other genes may be important forsignaling differentiation and would be expected to be tissue-specific oractivated only by a restricted family of similar ligands.

An additional means of elucidating the mechanisms of IL-2-mediatedtransmembrane signalling is provided by the varied effects of elevatedcAMP on IL-2-induced gene expression. The diverse responses of the genesto cAMP suggest that the IL-2 signalling pathways responsible for theirinduction must bifurcate at a point prior to intersection with the cAMPregulated pathways. One potential mechanism of cAMP action may involveregulation of protein phosphorylation, as cAMP is an activator ofprotein kinase A, and elevations of intracellular cAMP inhibitIL-2-inducted phosphorylation events. In addition, as cAMP blocksIL-2-stimulated cell cycle progression at a point in early G₁, cAMPsensitivity is a useful tool with which to dissect IL-2-mediated signaltransduction pathways involved in cell cycle progression.

A likely function of the immediate/early gene products is the governingof subsequent DNA replication and cell division. Previouslycharacterized IL-2 induced genes encode kinases (c-raf-1, pim-1) and DNAbinding proteins (c-fos, c-myc, c-myb). Further sequence analysis of thenovel genes will determine whether the proteins they encode containconserved domains which would implicate similar functions. However,since IL-2 stimulates cellular differentiation as well as division, andhas been shown to induce the expression of a number of genes which donot per se perform a direct role in cell cycle progression, a functionalcorrelation between the expression of the novel genes and cell cycletransit should be demonstration.

Indirectly, cAMP sensitivity is suggestive of involvement in G₁progression. The demonstration of induction of the genes by other growthfactors, as well as heightened expression in transformed cell lineswould further support this notion. A more direct approach, utilizingantisense oligonucleotides, will make it possible to determine whetherspecific blockage of expression of any of these genes is sufficient toprevent cell cycle progression. Similarly, it will be possible todetermine whether the immediate early gene products exert cell cyclecontrol through the induction of expression of late genes, as has beendemonstrated for regulation of the PCNA/cyclin, DNA polymerase A andcdc2 genes by the c-myb and c-myc gene products. Interestingly, theIL-2-induced expression of the PCNA/cyclin and DNA topoisomerase II genein late G₁ is specifically inhibited by cAMP, so that cAMP sensitivityof immediate early gene expression can provide a useful indicator ofwhich genes play a role in regulating late gene expression. If, like thepreviously characterized cell cycle regulatory cdc2/CDC28 and cyclingenes, the novel IL-2 induced genes are highly conserved, then it mayultimately be possible to isolate yeast homologs of the clones andperform deletional analyses to further define the functions of the geneproducts.

Ultimately, the definitive assignment of a given gene product to aparticular function within a cell depends upon a series of differentapproaches, including determining intracellular location, anddetermining the consequences of blocking the expression of the geneeither by mRNA antisense methods or by homologous recombination methods.All of the methods necessary for these studies exist as prior art andtherefore, given the identification of a given gene as activated by aligand such as the cytokine IL-2 is possible to characterize each geneproduct.

By following the method of the invention, eight clones containingligand-induced genes have been identified. At least six of theseligand-induced genes are novel and have been named Cytokine Response(CR) genes 1-3, 5, 6 and 8. CR4 is identical to a gene reported asSATB-1 (Dickinson, L. A. et al. (1992) Cell 70:631-645), for SpecialAT-rich Binding protein 1, which binds selectively to the nuclearmatrix/scaffold-associating region of DNA. CR7 is identical to theputative proto-oncogene, pim 1, a known IL-2-induced gene. The nucleicacid sequences of these CR genes, i.e., CR genes 1-6 and 8, are setforth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, and 13 (these sequences are alsoshown in FIGS. 1-7). The amino acid sequences encoded by these CR genesare set forth in SEQ ID NOs.: 2, 4, 6, 8, 10, 12, and 14 (thesesequences are also shown in FIGS. 1-7) as well as in SEQ ID NOs: 1, 3,5, 7, 9, 11, and 13. Table I provides several characteristics of the CRgenes.

                  TABLE 1    ______________________________________    CR GENE CHARACTERISTICS           Protein Size                     Homology   IL-2 Induction                                         cAMP Effect    CR Gene           (kDa)     (Identity) (x)      (±)    ______________________________________    1      22.2      G.sub.0 S8, B134                                24       -    2      6.6       --         7        -    3      88.0      Prosta-    22       -                     glandin R    4      83.9      (SATB1)    6        +    5      28.4      SH2        50       -    6      17.5      GADD45,    5        -                     MyD 118    7      34.4      (pim)      17       -    8      45.3      bHLH       7        +    ______________________________________

The regulatory region of all of the CR genes can be used to constructassays to identify the relevant cis-acting DNA response elements, thetrans-acting factors responsible for transcriptional activation leadingto CR gene expression, and the biochemical signalling for pathwaystriggered by IL-2 (the ligand) that activate the transcriptionalactivating factors. These assays can be used to identify novel agents ordrugs that either suppress or activate CR gene expression. Such novelagents or drugs can be used immunosuppressives, immunostimulants, oranti-cancer agents.

The immediate-early CR genes and gene products can be used to constructassays to determine which biochemical and molecular events, initiated bythe ligand-receptor stimulation, promote progression to the intermediateand late stages of cell cycle progression that are responsible for DNAsynthesis and replication. These assays can be used to identify novelagents and drugs that either suppress or promote these processes. Withthe capacity to generate large quantities of the CR gene products, thethree-dimensional structures of the products can be determined byconventional methods, such as x-ray crystallography and nuclear magneticresonance. From this information, novel agents or drugs can beidentified using computer analysis of the chemical structures, thatinteract with the CR gene product. These agents can then be developed astherapeutics.

The regulation of CR1 expression is notable in that it is rapidly andtransiently induced by IL-2, and mRNA expression is suppressed byelevated intracellular cAMP, which also suppresses IL-2-promoted G₁progression. There are already available pharmaceuticals that elevateintracellular cAMP, such as aminophylline and theophylline. Therefore,it is now possible to determine how these agents function to inhibit CR1expression and to identify novel agents that act similarly, but may haveparticular pharmacologic advantages.

The CR1 gene includes 2406 nucleotides (shown in SEQ ID NO:1 and FIG. 1)and encodes a protein of 202 amino acids (about 22 kDa) (shown in SEQ IDNO:2 and FIG. 1) that shares sequence homology to two other recentlyreported genes, GOS8 and BL-34, both of which are induced to high levelsof expression by mitogens. The nucleotide sequence of the CR1 gene isabout 58% homologous to the nucleotide sequence of the GOS8 gene(Siderovski, D. P. et al. (1994) DNA and Cell Biology 13:125-147), whichwas isolated from a PHA-induced T cell library. At the protein level,CR1 is about 51.2% homologous to GOS8. In addition, the nucleotidesequence of the CR1 gene is about 58% homologous to the nucleotidesequence of the BL34 gene (Hong, J. X. et al. (1993) J. Immunol.150:3895-3904), which was isolated from a Staph A-activated B cell cDNAlibrary. At the protein level, CR1 is about 48.0% homologous to BL34.The homology of CR1 with BL-34 is of particular interest, in that BL-34is expressed only by activated B cells, is preferentially expressed invivo by B cells in lymph node germinal centers, and is overexpressed inB cell malignancies. As predicted from its amino acid sequence whichcontains neither a hydrophobic leader sequence nor a transmembraneregion, CR1 is an intracellular protein. Also, the CR1 protein includesno sequences consistent with other functional motifs or domains, such asfound for DNA binding proteins, kinases, phosphatases, or linkermolecules. The sequences for the gene (SEQ. ID No: 1), protein (SEQ IDNo: 2), and protein coding region of the gene (SEQ. ID No: 27) for CR1,the underlined being the protein coding region of the gene, are providedbelow in Table II.

                                      TABLE II    __________________________________________________________________________    Full Sequenced DNA and Deduced Protein Sequence for CR1    __________________________________________________________________________    AACCCAACCGCAGTTGACTAGCACCTGCTACCGCGCCTTTGCTTCCTGGCGCACGCGGAG 60     ##STR1##     ##STR2##     ##STR3##     ##STR4##     ##STR5##     ##STR6##     ##STR7##     ##STR8##     ##STR9##     ##STR10##     ##STR11##     ##STR12##     ##STR13##     ##STR14##    TGAGTCTCCACGGCAGTGAGG 742    AAGCCAGCCGGGAAGAGAGGTTGAGTCACCCATCCCCGAGGTGGCTGCCCCTGTGTGGGA 802    GGCAGGTTCTGCAAAGCAAGTGCAAGAGGACAAAAAAAAAAAAAAAAAAAAAAAATGCGC 862    TCCAGCAGCCTGTTTGGGAAGCAGCAGTCTCTCCTTCAGATACTGTGGGACTCATGCTGG 922    AGAGGAGCCGCCCACTTCCAGGACCTGTGAATAAGGGCTAATGATGAGGGTTGGTGGGGC 982    TCTCTGTGGGGCAAAAAGGTGGTATGGGGGTTAGCACTGGCTCTCGTTCTCACCGGAGAA1042    GGAAGTGTTCTAGTGTGGTTTAGGAAACATGTGGATAAAGGGAACCATGAAAATGAGAGG1102    AGGAAAGACATCCAGATCAGCTGTTTTGCCTGTTGCTCAGTTGACTCTGATTGCATCCTG1162    TTTTCCTAATTCCCAGACTGTTCTGGGCACGGAAGGGACCCTGGATGTGGAGTCTTCCCC1222    TTTGGCCCTCCTCACTGGCCTCTGGGCTAGCCCAGAGTCCCTTAGCTTGTACCTCGTAAC1282    ACTCCTGTGTGTCTGTCCAGCCTTGCAGTCATGTCAAGGCCAGCAAGCTGATGTGACTCT1342    TGCCCCATGCGAGATATTTATACCTCAAACACTGGCCTGTGAGCCCTTTCCAAGTCAGTG1402    GAGAGCCCTGAAAGGAGCCTCACTTGAATCCAGCTCAGTGCTCTGGGTGGCCCCCTGCAG1462    GTGCCCCCTGACCCTGCGTTGCAGCAGGGTCCACCTGTGAGCAGGCCCGCCCTGGGCCCT1522    CTTCCTGGATGTGCCCTCTCTGAGTTCTGTGCTGTCTCTTGGAGGCAGGGCCCAGGAGAA1582    CAAAGTGTGGAGGCCTCGGGGAGTGACTTTTCCAGCTCTCATGCCCCGCAGTGTGGAACA1642    AGGCAGAAAAGGATCCTAGGAAATAAGTCTCTTGGCGGTCCCTGAGAGTCCTGCTGAAAT1702    CCAGCCAGTGTTTTTTGTGGTATGAGAACAGCCAAAAAGAGATGCCCCGAGATAGAAGGG1762    GAGCCTTGTGTTTCTTTCCTGCAGACGTGAGATGAACACTGGAGTGGGCAGAGGTGGCCC1822    AGGACCATGACACCCTTAGAGTGCAGAAGCTGGGGGGAGAGGCTGCTTCGAAGGGCAGGA1882    CTGGGGATAATCAGAACCTGCCTGTCACCTCAGGGCATCACTGAACAAACATTTCCTGAT1942    GGGAACTCCTGCGGCAGAGCCCAGGCTGGGGAAGTGAACTACCCAGGGCAGCCCCTTTGT2002    GGCCCAGGATAATCAACACTGTTCTCTCTGTACCATGAGCTCCTCCAGGAGATTATTTAA2062    GTGTATTGTATCATTGGTTTTCTGTGATTGTCATAACATTGTTTTTGTTACTGTTGGTGC2122    TGTTGTTATTTATTATTGTAATTTCAGTTTGCCTCTACTGGAGAATCTCAGCAGGGGTTT2182    CAGCCTGACTGTCTCCCTTTCTCTACCAGACTCTACCTCTGAATGTGCTGGGAACCTCTT2242    GGAGCCTGTCAGGAACTCCTCACTGTTTAAATATTTAGGTATTGTGACAAATGGAGCTGG2302    TTTCCTAGAAATGAATGATGTTTGCAATCCCCATTTTCCTGTTTCAGCATGTTATATTCT2362    TATGAAATAAAAGCCCAAGTCCAATATGAAAAAAAAAAAAAAAA(SEQ. ID No:    __________________________________________________________________________    1)2406

The CR2 gene includes 1283 nucleotides (shown in SEQ ID NO:3 and FIG. 2)and encodes a small, intracellular protein of 60 amino acids (about 6.6kDa)(shown in SEQ ID NO:4 and FIG. 2). The CR2 gene is the only CR genefor which there are no homologies to known gene products. Elevated cAMPsuppresses, but does not abolish CR2 gene expression. The sequences forthe gene (SEQ. ID No: 3), protein (SEQ. ID No: 4), and protein codingregion of the gene (SEQ ID No: 28) for CR2, the underlined being theprotein coding region of the gene (SEQ. ID No: 28), are provided belowin Table III.

                                      TABLE III    __________________________________________________________________________    Full DNA Sequence and Deduced Protein Sequence for CR2    __________________________________________________________________________    ATTTAGAGCAACTCAGGAAATAGGTGCACACAAGCAAACCATGTGGTTAAAGCCTTTGGA 60    ACTGGTTTGAGCAAAGCTGTAGGTGATTTGACAAAATCATCTGCAAAACCAGATTTCTAA120     ##STR15##     ##STR16##     ##STR17##     ##STR18##     ##STR19##    TGACTTACCAGTTTTACTTTC371    AGTCTCTCCATTTCTAATTAAATGAGATGCAGAAATGCTGGTGCCTTGCTATGATGTTTG431    CAGTTATTATTTCTAGGAAAAAAAATATTATTGTTACTCAGTATCTGGTCTAGCTACTTG491    GACAACTGGACTATCCCCCTCCTTTCAAGGGAGGGCAAAGCATTTCAGAAAAGAACTAAG551    TGCTATTTCTCTGCTTCAGGAATGTCTCCCGTATGTAAAAGAATGTGGCTTCAGGGAGTA611    GCATGTGTTGTAAAGGTGGATGGGTCTAACTTCATGGACAGCTCTGACATCCACTAGCTA671    TGCCACCTGATGCAAACCACTTGGGCTGTCTGCAGTTTCGTTTATCTTTCTGGAATTGGT731    AATAACAACCACCTGGCAAGATCACTGTTATGAATACGGAGGATCAAAGTTGTGAAGTTA791    TTTTGTAAAGTGAAATGTTCTGAAAAATGGATTTTAACAGTGTCAGCGAAAAGTAGATTT851    TTGACATTTATCAAGAGTTCAGCTAATGAAAACAAGTATGGATAATAGTTACATAGAACT911    GTCTACTTTACTCAGTACTTTAGCATATGCTATTATATTTAATCTTCTTAAAAAGTAGGA971    AATTATACAAGCCATGTATTGATATTATTGTGGTGGTTGTCGTTCTCAATTACACACTGA1031    ATATTAAGACCTCTCAGGTAGCAGCTGGAAGGACATTGTATCCAGTTTCCTGATTGTTTT1091    CAATGGAATAATCATGTATACATGCACTACTAATGAGACAATGGTGATTCTAAAAGCTTA1151    ATCAGGGGGACTTTTGTGTATTCCAAATCTACTAAAAATAAAGAAACACAGAAATGAGAA1211    AAAAAAAAAAAA(SEQ. ID No: 3)1223    __________________________________________________________________________

The CR3 gene includes 2451 nucleotides (shown in SEQ ID NO:5 and FIG. 3)and encodes a protein of 378 amino acids (about 41.5 kDa) (shown in SEQID NO:6 and FIG. 3). This protein is homologous to G-coupled, 7transmembrane-spanning receptors of the prostaglandin family. Thereceptor for prostacyclin (PGI₂) is most homologous (about 70%) (SeeBoie, Y. et al. (1994) J. Biol. Chem. 269:12173-12178) to the CR3protein. PGI₂ is a labile metabolite of arachidonic acid produced viathe cyclooxygenase pathway, and plays a major physiological role as apotent mediator of vasodilation and inhibitor of platelet activation. Itis primarily expressed in the kidney with lower levels of mRNA alsoobserved in the lung and the liver. In the kidney the PGI₂ receptor isthought to play an important role in renal blood flow, renin release,and glomerular filtration rate. By comparison, CR3 is maximallyexpressed by leukocytes, placenta, testes, ovary and small intestine,and at lower levels by spleen, thymus and prostate, but not by kidney orliver. Therefore, CR3 most likely plays a regulatory role in cellularproliferation and/or inflammation. Elevated cAMP suppresses CR3expression early on after IL-2 stimulation, but not at later timeintervals.

Because the CR3 encodes a protein that is a member of a family of 7transmembrane spanning receptors, it is likely that this receptor iscoupled to cytoplasmic GTP-binding proteins (G proteins) that are knownto activate or suppress the generation of cAMP. Therefore, the CR3 geneproduct provides a new receptor that can allow the manipulation ofcellular functions that are controlled by biochemical pathways signaledby the receptor. The CR3 gene and gene product can be used in assays foridentifying ligands that trigger the receptor. These ligands can be usedto modulate cellular proliferation and inflammation. The sequences forthe gene (SEQ. ID No: 5), protein (SEQ. ID No: 6), and protein codingregion of the gene (SEQ. ID No: 29) corresponding to CR3, the underlinedbeing the protein coding region of the gene (SEQ. ID No: 29), are shownin Table IV below.

                                      TABLE IV    __________________________________________________________________________    Full DNA and Protein Sequences for CR3    __________________________________________________________________________    CGCGGGAGCCTCGAGCGCCGCTCGGATGCAGAAGCCGAGCCGCCACTCGGCGCGCGGTGG 60    GAGACCCAGGGCAAGCCGCCGTCGGCGCGCTGGGTGCGGGAAGGGGGCTCTGGATTTCGG 120    TCCCTCCCCTTTTTCCTCTGAGTCTCGGAACGCTCCAGATCTCAGACCCTCTTCCTCCCA 180     ##STR20##     ##STR21##     ##STR22##     ##STR23##     ##STR24##     ##STR25##     ##STR26##     ##STR27##     ##STR28##     ##STR29##     ##STR30##     ##STR31##     ##STR32##     ##STR33##     ##STR34##     ##STR35##     ##STR36##     ##STR37##     ##STR38##     ##STR39##     ##STR40##     ##STR41##     ##STR42##     ##STR43##    TGAGGTCAGTAGTTTAAAAGTTCTTAGTTATATAGCATCTG1343    GAAGATCATTTTGAAATTGTTCCTTGGAGAAATGAAAACAGTGTGTAAACAAAATGAAGC1403    TGCCCTAATAAAAAGGAGTATACAAACATTTAAGCTGTGGTCAAGGCTACAGATGTGCTG1463    ACAAGGCACTTCATGTAAAGTGTCAGAAGGAGCTACAAAACCTACCCTCAGTGAGCATGG1523    TACTTGGCCTTTGGAGGAACAATCGGCTGCATTGAAGATCCAGCTGCCTATTGATTTAAG1583    CTTTCCTGTTGAATGACAAAGTATGTGGTTTTGTAATTTGTTTGAAACCCCAAACAGTGA1643    CTGTACTTTCTATTTTAATCTTGCTAGTACCGTTATACACATATAGTGTACAGCCAGACC1703    AGATTAAACTTCATATGTAATCTCTAGGAAGTCAATATGTGGAAGCAACCAAGCCTGCTG1763    TCTTGTGATCACTTAGCGAACCCTTTATTTGAACAATGAAGTTGAAAATCATAGGCACCT1823    TTTACTGTGATGTTTGTGTATGTGGGAGTACTCTCATCACTACAGTATTACTCTTACAAG1883    AGTGGACTCAGTGGGTTAACATCAGTTTTGTTTACTCATCCTCCAGGAACTGCAGGTCAA1943    GTTGTCAGGTTATTTATTTTATAATGTCCATATGCTAATAGTGATCAAGAAGACTTTAGG2003    AATGGTTCTCTCAACAAGAAATAATAGAAATGTCTCAAGGCAGTTAATTCTCATTAATAC2063    TCTTTATCCTATTTCTGGGGGAGGATGTACGTGGCCATGTATGAAGCCAAATATTAGGCT2123    TAAAAACTGAAAAATCTGGTTCATTCTTCAGATATACTGGAACCCTTTTAAAGTTGATAT2183    TGGGGCCATGAGTAAAATAGATTTTATAAGATGACTGTGTTGTACTAAAATTCATCTGTC2243    TATATTTTATTTAGGGGACATGGTTTGACTCATCTTATATGGGAAACCATGTAGCAGTGA2303    GTCATATCTTAATATATTTCTAAATGTTTGGCATGTAAACGTAAACTCAGCATCACAATA2363    TTTCAGTGAATTTGCACTGTTTAATCATAGTTACTGTGTAAACTCATCTGAAATGTTACC2423    AAAAATAAACTATAAAACAAAATTTGA(SEQ ID No: 5)2450    __________________________________________________________________________

The CR4 gene includes 2946 nucleotides (shown in SEQ ID NO:7 and FIG. 4)and encodes a protein of 763 amino acids (about 85.9 kDa) (shown in SEQID NO:8 and FIG. 4). The sequence of this gene is identical to a genereported as SATB-1 (Dickinson, L. A. et al. (1992) Cell 70:631-645), forSpecial AT-rich Binding protein 1, which binds selectively to thenuclear matrix/scaffold-associating region of DNA. It is expressedexclusively in the thymus and activated peripheral T cells. CR4 is theonly CR gene also activated by the TCR. In addition, elevated cAMPactually stimulates CR4 gene expression.

Because the CR4 gene product binds to special AT-rich regions of DNAknown to associate with proteins in the nuclear mating, CR4 is mostlikely a novel nuclear matrix protein. The nuclear matrix proteins areknown to influence the structure of DNA, facilitating transcription ofspecific genes in particular differentiated tissues. Because theexpression of CR4 is restricted to thymocytes and activated T cells, itis likely that CR4 plays an important role in T cell maturation,differentiation or proliferation. Therefore, novel agents that modifyCR4 gene expression or CR4 function have the potential to be useful tomanipulate the T cell immune response. Thus, CR4 can be used in an assayto identify such novel agents which can be used, for example, to treattransplant recipients by, for example, inhibiting the recipient's T cellimmune response. These agents can also be used to stimulate the T cellimmune response in immunosuppressed subjects, e.g., AIDS patients. Thesequences for the gene (SEQ. ID No: 7), protein (SEQ. ID No: 8), andprotein coding region of the gene (SEQ. ID No: 30) for CR4, theunderlined being the protein coding region of the gene, are shown inTable V below.

                                      TABLE V    __________________________________________________________________________    Full DNA and Deduced Protein Sequence for CR4    __________________________________________________________________________    GGGGGGAAAGGAAAATAATACAATTTCAGGGGAAGTCGCCTTCAGGTCTGCTGCTTTTTT 60    ATTTTTTTTTTTTTAATTAAAAAAAAAAAGGACATAGAAAACATCAGTCTTGAACTTCTC 120    TTCAAGAACCCGGGCTGCAAAGGAAATCTCCTTTGTTTTTGTTATTTATGTGCTGTCAAG 180     ##STR44##     ##STR45##     ##STR46##     ##STR47##     ##STR48##     ##STR49##     ##STR50##     ##STR51##     ##STR52##     ##STR53##     ##STR54##     ##STR55##     ##STR56##     ##STR57##     ##STR58##     ##STR59##     ##STR60##     ##STR61##     ##STR62##     ##STR63##     ##STR64##     ##STR65##     ##STR66##     ##STR67##     ##STR68##     ##STR69##     ##STR70##     ##STR71##     ##STR72##     ##STR73##     ##STR74##     ##STR75##     ##STR76##     ##STR77##     ##STR78##     ##STR79##     ##STR80##     ##STR81##     ##STR82##     ##STR83##     ##STR84##     ##STR85##     ##STR86##     ##STR87##     ##STR88##     ##STR89##     ##STR90##     ##STR91##     ##STR92##    TGAGATAAAAGTATTTGTTTCGTTCAACAGTGCCACTGGT2543    ATTTACTAACAAAATGAAAAGTCCACCTTGTCTTCTCTCAGAAAACCTTTGTTGTTCATT2603    GTTTGGCCAATGAACTTTCAAAAACTTGCACAAACAGAAAAGTTGGAAAAGGATAATACA2663    GACTGCACTAAATGTTTTCCTCTGTTTTACAAACTGCTTGGCAGCCCCAGGTGAAGCATC2723    AAGGATTGTTTGGTATTAAAATTTGTGTTCACGGGATGCACCAAAGTGTGTACCCCGTAA2783    GCATGAAACCAGTGTTTTTTGTTTTTTTTTTAGTTCTTATTCCGGAGCCTCAAACAAGCA2843    TTATACCTTCTGTGATTATGATTTCCTCTCCTATAATTATTTCTGTAGCACTCCACACTG2903    ATCTTTGGAAACTTGCCCCTTATTTAAAAAAAAAAAAAAAAAA(SEQ. ID No:    __________________________________________________________________________    7)2946

The CR5 gene includes 2020 nucleotides (shown in SEQ ID NO:9 and FIG. 5)and encodes a protein of 258 amino acids (about 28 kDa) (shown in SEQ IDNO:10 and FIG. 5). In the middle of the open reading frame of the CR5protein is about a 100 amino acid region that has sequence homology(about 25-35%) to src homology 2 (SH2) domains (Waksman, G. et al.(1993) Cell 72:779-790), found in many proteins that bind tophosphotyrosine residues, e.g., kinases, substrates, linking molecules,and transcription factors. On either side of this SH2 domain the aminoacid sequence is very rich in proline residues. Analysis of CR5 proteinexpression by different tissues reveals a high level of expression inheart, placenta, lung, liver skeletal muscle and kidney. CR5 proteinexpression is induced by the proliferation-promoting cytokines IL-2,IL-3, IL-4, IL-5, but not by IL-6. Also, CR5 protein expression isinduced by IFN-β and elevated intracellular cAMP, both of whichantagonize IL-2 promoted proliferation. CR5 protein has been found tointeract with a subunit of the RNA polymerase II preinitiation complex,termed RNA polymerase II elongation factor SIII, p15 subunit. Garret, K.P. et al. (1994) Proc. Natl. Acad. Sci. USA 91:5237-5241. The p15subunit of this RNA polymerase II elongation factor is known to beresponsible for promoting the elongation of transcripted mRNA molecules.Therefore, CR5 appears to function as a ligand-stimulated factor thatfacilitates mRNA expression by promoting the full elongation of mRNAtranscripts. This phenomenon promises to be a novel way in whichligand-receptor systems can regularly promote gene expression.Previously, attention has focused almost entirely on this initiation oftranscription, not the elongation of transcripts that were prematurelytruncated. Accordingly, novel agents or drugs that modify CR5 geneexpression or CR5 function have the potential to provide new ways toalter ligand-stimulated gene expression and thereby alter cellularfunction. The sequences for the gene (SEQ. ID No: 9), protein (SEQ. IDNo: 10), and protein coding region of the gene (SEQ. ID No: 33) for CR5,the underlined being the protein coding region of the gene (SEQ. ID No:33), are shown in Table VI below.

                                      TABLE VI    __________________________________________________________________________    Full DNA and Deduced Protein Sequence for CR5    __________________________________________________________________________    CGCCCGCGCGCCCCGGGAGCCTACCCAGCACGCGCTCCGCGCCCACTGGTTCCCTCCAGC 60     ##STR93##     ##STR94##     ##STR95##     ##STR96##     ##STR97##     ##STR98##     ##STR99##     ##STR100##     ##STR101##     ##STR102##     ##STR103##     ##STR104##     ##STR105##     ##STR106##     ##STR107##     ##STR108##     ##STR109##    GACTGTACGGGGCAATCTGCCCACCCTCACCCAGTCGCACCCTGGAGGGGACATCAGCCC 946    CAGCTGGACTTGGGCCCCCACTGTCCCTCCTCCAGGCATCCTGGTGCCTGCATACCTCTG1006    GCAGCTGGCCCAGGAAGAGCCAGCAAGAGCAAGGCATGGGAGAGGGGAGGTGTCACACAA1066    CTTGGAGGTAAATGCCCCCAGGCCGCATGTGGCTTCATTATACTGAGCCATGTGTCAGAG1126    GATGGGGAGACAGGCAGGACCTTGTCTCACCTGTGGGCTGGGCCCAGACCTCCACTCGCT1186    TGCCTGCCCTGGCCACCTGAACTGTATGGGCACTCTCAGCCCTGGTTTTTCAATCCCCAG1246    GGTCGGGTAGGACCCCTACTGGCAGCCAGCCTCTGTTTCTGGGAGGATGACATGCAGAGG1306    AACTGAGATCGACAGTGACTAGTGACCCCTTGTTGAGGGGTAAGCCAGGCTAGGGGACTG1366    CACAATTATACACTCCTGAGCCCTGGTAGTCCAGAGACCCCAACTCTGCCCTGGCTTCTC1426    TGGTTCTTCCCTGTGGAAAGCCCATCCTGAGACATCTTGCTGGAACCAAGGCAATCCTGG1486    ATGTCCTGGTACTGACCCACCCGTCTGTGAATGTGTCCACTCTCTTCTGCCCCCAGCCAT1546    ATTTGGGGAGGATGGACAACTACAATAGGTAAGAAAATGCAGCCGGAGCCTCAGTCCCCA1606    GCAGAGCCTGTGTCTCACCCCCTCACAGGACAGAGCTGTATCTGCATAGAGCTGGTCTCA1666    CTGTGGCGCAGGCCCCGGGGGGAGTGCCTGTGCTGTCAGGAAGAGGGGGTGCTGGTTTGA1726    GGGCCACCACTGCAGTTCTGCTAGGTCTGCTTCCTGCCCAGGAAGGTGCCTGCACATGAG1786    AGGAGAGAAATACACGTCTGATAAGACTTCATGAAATAATAATTATAGCAAAGAACAGTT1846    TGGTGGTCTTTTCTCTTCCACTGATTTTTCTGTAATGACATTATACCTTTATTACCTCTT1906     ##STR110##    __________________________________________________________________________

The CR6 gene includes 1066 nucleotides (shown in SEQ ID NO:11 and FIG.6) and encodes protein of 159 amino acids (about 17.5 kDa) (shown in SEQID NO:12 and FIG. 6). This gene belongs to a family of smallnuclear-localizing gene products. Two other members of this family,GADD45 and MyD118, have been identified. GADD45 was cloned from humanfibroblasts induced by UV irradiation (Papathanasiou, M. A. et al.(1991) Mol. Cell Biol. 11(2):1009-1016). This protein is regulated byp53 and suppresses growth of cells by binding to PCNA, a co-factorrequired for DNA polymerase δ activity. (Smith, M. L. et al. (1994)Science 266:1376-1380). MyD118 was cloned from M1D+ myeloid precursorsfollowing induction of terminal differentiation and growth arrest byIL6. Abdollahi, A. et al. (1991) Oncogene 6:165-167. At the nucleotidelevel, CR6 is about 65% homologous to GADD45. At the protein level, CR6is about 54% homologous to GADD45. At the nucleotide level, CR6 is about66% homologous to MyD118. At the protein level, CR6 is about 53%homologous to MyD118. The CR6 protein is expressed only in testes, ovaryand prostate, and its expression is suppressed by elevated cAMP.

By analogy to it's homology to GADD45 and MyD118, the CR6 gene productmost likely plays a role in DNA replication. Thus far, experiments haveindicated that CR6 expression is not induced by agents that damage DNA,such as UV light. Moreover, CR6 does not bind to PCNA. However, CR6 doespromote DNA replication in vitro, and it is likely to be a novelCD-factor necessary for DNA replication. Therefore, the CR6 gene productcan be used to identify inhibitors of DNA replication which can be usedas anti-proliferative agents, e.g., in the treatment of cancer. The SEQ.ID No: 11, SEQ. ID No: 12, and SEQ. ID No: 31, corresponding to thegene, protein and protein coding DNA sequences of CR6, the underlinedcorresponding to the protein coding region of the gene (SEQ. ID No: 31),are shown in Table VII below.

                                      TABLE VII    __________________________________________________________________________    Full DNA and Deduced Protein Sequence for CR6    __________________________________________________________________________    GTGGGTGCGCCGTGCTGAGCTCTGGCTGTCAGTGTGTTCGCCCGCGTCCCCTCCGCGCTC 60     ##STR111##     ##STR112##     ##STR113##     ##STR114##     ##STR115##     ##STR116##     ##STR117##     ##STR118##     ##STR119##     ##STR120##     ##STR121##    TGACAGCCCGGCGGGGACCTT 595    GGTCTGATCGACGTGGTGACGCCCCGGGGCGCCTAGAGCGCGGCTGGCTCTGTGGAGGGG 655    CCCTCCGAGGGTGCCCGAGTGCGGCGTGGAGACTGGCAGGCGGGGGGGGCGCCTGGAGAG 715    CGAGGAGGCGCGGCCTCCCGAGGAGGGGCCCGGTGGCGGCAGGGCCAGGCTGGTCCGAGC 775    TGAGGACTCTGCAAGTGTCTGGAGCGGCTGCTCGCCCAGGAAGGCCTAGGCTAGGACGTT 835    GGCCTCAGGGCCAGGAAGGACAGACTGGCCGGGCAGGCGTGACTCAGCAGCCTGCGCTCG 895    GCAGGAAGGAGCGGCGCCCTGGACTTGGTACAGTTTCAGGAGCGTGAAGGACTTAACCGA 955    CTGCCGCTGCTTTTTCAAAACGGATCCGGGCAATGCTTCGTTTTCTAAAGGATGCTGCTG1015     ##STR122##    __________________________________________________________________________

The CR7 gene includes 2400 nucleotides and encodes a protein of 313amino acids (about 34 kDa). The CR7 gene is identical to the putativeproto-oncogene, pim-1, which has been reported to be over-expressed inabout 50% of Moloney murine leukemia virus (MuLV)-induced T celllymphomas. See Selten, G. et al. (1986) Cell 46:603-611 for thenucleotide and amino acid sequence of pim-1) Pim-1 is known to be anIL-2-induced gene and is a serine/threonine-specific protein kinaseinvolved in T cell lymphomagenesis. The CR7 gene includes a 2400nucleotide DNA (SEQ. ID No: 25), encoding a protein of 313 amino acids(about 34 kD) of SEQ. ID No: 26. The gene (SEQ. ID No: 25), protein(SEQ. ID No: 26), protein coding region of the gene (SEQ. ID No: 35),and the DNA sequence complementary to the gene sequence (SEQ. ID No:34), the underlined being the protein coding region of the gene, areshown in Table VIII below.

                                      TABLE VIII    __________________________________________________________________________    Full DNA Sequence and Protein Sequence for CR7    __________________________________________________________________________     ##STR123##     ##STR124##     ##STR125##     ##STR126##     ##STR127##     ##STR128##     ##STR129##     ##STR130##     ##STR131##     ##STR132##     ##STR133##     ##STR134##     ##STR135##     ##STR136##     ##STR137##     ##STR138##     ##STR139##     ##STR140##     ##STR141##     ##STR142##     ##STR143##     ##STR144##     ##STR145##     ##STR146##     ##STR147##     ##STR148##     ##STR149##     ##STR150##     ##STR151##     ##STR152##     ##STR153##     ##STR154##     ##STR155##     ##STR156##     ##STR157##     ##STR158##     ##STR159##     ##STR160##     ##STR161##     ##STR162##     ##STR163##     ##STR164##     ##STR165##    __________________________________________________________________________

The CR8 gene includes 2980 nucleotides (shown in SEQ ID NO:13 and FIG.7) and encodes (via a 3.2 kb mRNA transcript) a protein of 412 aminoacids (about 45 kDa) (shown in SEQ ID NO:14 and FIG. 7). There issignificant sequence homology (40-45%) within an N-terminal 58 aminoacid residue region to transcription factors that have abasic-Helix-Loop-Helix (bHLH) motif. The protein encoded by the bHLHregion of the gene has been expressed in E. coli and has been found tobind to a hexanucleotide predicted by the binding specificity of otherbHLH proteins. See Feder, J. et al. (1993) Mol. Cell Biol.13(1):105-113. The N-terminal basic region binds to DNA and the HLHregion serves as a protein dimerization motif. From the sequence of thebHLH region, CR8 fits into a class by itself. It shares most homologywith Drosophila transcription repressors of the hairy family. However,CR8 lacks amino acid residues in the basic region and a C-terminal WRPWmotif, characteristic for hairy proteins. CR8 also binds to Class BE-box sites (CACGTC/CATGTG), as do the c-myc family of bHLH proteins,rather than to Class C sites (CAGCCG) preferred by hairy-related familymembers. CR8 is ubiquitously expressed in all tissues examined exceptplacenta. Its expression is induced by cytokines such as IL-2 and IL-3,which stimulate cellular proliferation, and also by IFNβ and elevatedcAMP, which antagonize proliferation.

Because CR8 contains a bHLH domain, it is most likely a protein thatbinds to DNA and modifies gene expression, either by activation or bysuppression. Since CR8 binds to class B E-bax sequences, which theproto-oncogene c-myc family members also bind, it is likely that CR8modifies the expression of genes important for the intermediate and latephases of ligand-promoted cell cycle progression. It follows that CR8 isa prime candidate for the development of new assays to discover agentsthat modify cellular function by either enhancing or suppressing CR8gene expression or CR8 function. The CR8 gene and its gene product aredescribed in further detail below in Example VII. The CR8 gene includesa 2980 nucleotide fragment of SEQ. ID NO: 13, which encodes (via a 3.2Kb mRNA transcript) a protein of 412 amino acids (about 45 kD) of SEQ.ID NO: 14. The SEQ. ID No: 13, SEQ. ID No: 14, and SEQ. ID No: 32,corresponding to the gene, protein, and protein coding gene sequences,are shown in Table IX below.

                                      TABLE IX    __________________________________________________________________________    Full DNA and Deduced Protein Sequence for CR8    __________________________________________________________________________    CACACCGCCAGTCTGTGCGCTGAGTCGGAGCCAGAGGCCGCGGGGACACCGGGCCATGCA 60    CGCCCCCAACTGAAGCTGCATCTCAAAGCCGAAGATTCCAGCAGCCCAGGGGATTTCAAA 120    GAGCTCAGACTCAGAGGAACATCTGCGGAGAGACCCCCGAAGCCCTCTCCAGGGCAGTCC 180    TCATCCAGACGCTCCGTTAGTGCAGACAGGAGCGCGCAGTGGCCCCGGCTCGCCGCGCC 239     ##STR166##     ##STR167##     ##STR168##     ##STR169##     ##STR170##     ##STR171##     ##STR172##     ##STR173##     ##STR174##     ##STR175##     ##STR176##     ##STR177##     ##STR178##     ##STR179##     ##STR180##     ##STR181##     ##STR182##     ##STR183##     ##STR184##     ##STR185##     ##STR186##     ##STR187##     ##STR188##     ##STR189##     ##STR190##     ##STR191##    TAAACTCTCTA1486    GGGGATCCTGCTGCTTNGCTTTCCTNCCTCGCTACTTCCTAAAAAGCAACCNNAAAGNTT1546    TNGTGAATGCTGNNAGANTGTTGCATTGTGTATACTGAGATAATCTGAGGCATGGAGAGC1606    AGANNCAGGGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTATGTGCGTGTGCGTGCACA1666    TGTGTGCCTGCGTGTTGGTATAGGACTTTANNGCTCCTTNNGGCATAGGGAAGTCACGAA1726    GGATTGCTNGACATCAGGAGACTNGGGGGGGATTGTAGCACACGTCTGGGCTTNNCCCCA1786    CCCAGAGAATAGCCCCCNNCNANACANATCAGCTGGATTTACAAAAGCTTCAAAGTCTTG1846    GTCTGTGAGTCACTCTTCAGTTTGGGAGCTGGGTCTGTGGCTTTGATCAGAAGGTACTTT1906    CAAAAGAGGGCTTTCCAGGGCTCAGCTCCCAACCAGCTGTTAGGACCCCACCCTTTTGCC1966    TTTATTGTCGACGTGACTCACCAGACGTCGGGGAGAGAGAGCAGTCAGACCGAGCTTTTC2026    TGCTAACATGGGGAGGGTAGCAGACACTGGCATAGCACGGTAGTGGTTTGGGGGAGGGTT2086    TCCGCAGGTCTGCTCCCCACCCCTGCCTCGGAAGAATAAAGAGAATGTAGTTCCCTACTC2146    AGGCTTTCGTAGTGATTAGCTTACTAAGGAACTGAAAATGGGCCCCTTGTACAAGCTGAG2206    CTGCCCCGGAGGGAGGGAGGAGTTCCCTGGGCTTCTGGCACCTGTTTCTAGGCCTAACCA2266    TTAGTACTTACTGTGCAGGGAACCAAACCAAGGTCTGAGAAATGCGGACANCCCGAGCGA2326    GCACCCCAAAGTGCACAAAGCTGAGTAAAAAGCTGCCCCCTTCAAACAGAACTAGACTCA2386    GTTTTCAATTCCATCCTAAAACTCCTTTTAACCAAGCTTAGCTTCTCAAAGGGCTAACCA2446    AGCCTTGGAACGGCCAGATCCTTTCTGTAGGCTAATTCCTCTTGGCCAACGGCATATGGA2506    GTGTCCTTATTGCTAAAAAGGATTCCGNCTCCTTCAAAGAAGTTTTATTTTTGGTCCAGA2566    GTACTTGTTTTCCCGATGTGTCCAGCCAGCTCCGCAGCAGCTTTTCAAAATGCACTATGC2626    CTGATTGCTGATCGTGTTTTAACTTTTTCTTTTCCTGTTTTTATTTTGGTATTAAGTCGC2686    TGGCTTTATTTGTAAAGCTGTTATAAATATATATTATATNAANTATATTAAAAAGGAAAN2746    TGTTNCAGATGTTTATTTGTATAATTACTTGATTCACANAGNGAGAAAAANTGANTGTAT2806    TCCTGTNTTNGAAGAGAAGANNAATTTTTTTTTTCTCTAGGGAGAGGTACAGNGTTNNTN2866    TTTTGGGGCCTNCCNGAAGGGGTAAANNNGAAAATNTTTCTATNTATGAGTAAATGTTAA2926     ##STR192##    __________________________________________________________________________

In summary, of the eight CR genes isolated using the thiol-selectedIL-2-induced cDNA library, two are DNA binding proteins, one is a newlyrecognized transmembrane receptor, one contains an SH2 domain, one ishomologous to a newly recognized family of small proteins that regulatecellular proliferation, and another is a serine/threonine kinase alreadyknown to be IL-2-induced, and to be over-expressed in MuLV-induced Tcell lymphomas. Allowing for redundancies, a conservative estimate isthat there are still about 40-50 novel genes induced by IL-2 which canbe isolated using the method of the present invention.

Accordingly, the present invention pertains to an isolated nucleic acidcomprising the nucleotide sequence encoding one of the subject CRproteins, e.g., CR1, CR2, CR3, CR4, CR5, CR6, and CR8, and/orequivalents of such nucleic acids. The term "nucleic acid" as usedherein is intended to include fragments and equivalents. The term"equivalent" as used herein refers to nucleotide sequences encodingfunctionally equivalent CR proteins or functionally equivalent peptideswhich retain other activities of an CR protein such as described herein.Equivalent nucleotide sequences include sequences that differ by one ormore nucleotide substitutions, additions or deletions, such as allelicvariants; and, therefore, include sequences that differ from thenucleotide sequence CR proteins shown in any of SEQ ID NOs:2, 4, 6, 8,10, 12, and 14 due to the degeneracy of the genetic code. Equivalentsalso include nucleotide sequences that hybridize under stringentconditions (i.e., equivalent to about 20°-27° C. below the meltingtemperature (T_(m)) of the DNA duplex formed in about 1M salt) to thenucleotide sequence of the presently claimed CR proteins represented inSEQ ID NOs:2, 4, 6, 8, 10, 12, and 14. In one embodiment, equivalentsfurther include nucleic acid sequences derived from and evolutionarilyrelated to, a nucleotide sequences shown in any of SEQ ID NOs:1, 3, 5,7, 9, 11, and 13. Moreover, it is explicitly contemplated by the presentinvention that, under certain circumstances, it may be advantageous toprovide homologs of the subject CR proteins which have at least onebiological activity of a CR protein. Such homologs of the subject CRproteins can be generated by mutagenesis, such as by discrete pointmutation(s) or by truncation. For instance, mutation can give rise tohomologs which retain substantially the same, or merely a subset, of thebiological activity of the CR protein from which it was derived.Alternatively, antagonistic forms of the protein can be generated whichare able to inhibit the function of the naturally occurring form of theCR protein.

A protein has CR biological activity if it has one or more of thefollowing properties: (1) its expression is regulated by ligand-receptorstimulation; and (2) it participates in ligand-receptor modification ofcellular function, e.g. proliferation, differentiation, programmed celldeath.

As used herein, the term "gene" or "recombinant gene" refers to anucleic acid comprising an open reading frame encoding a CR protein ofthe present invention, including both exon and (optionally) intronsequences. A "recombinant gene" refers to nucleic acid encoding a CRprotein and comprising CR encoding exon sequences, though it mayoptionally include intron sequences which are either derived from achromosomal CR gene or from an unrelated chromosomal gene. The term"intron" refers to a DNA sequence present in a given CR gene which isnot translated into protein and is generally found between exons.

As used herein, the term "transfection" means the introduction of anucleic acid, e.g., an expression vector, into a recipient cell bynucleic acid-mediated gene transfer. "Transformation", as used herein,refers to a process in which a cell's genotype is changed as a result ofthe cellular uptake of exogenous DNA or RNA, and, for example, thetransformed cell expresses a recombinant form of the CR protein of thepresent invention or where anti-sense expression occurs from thetransferred gene, the expression of a naturally-occurring form of the CRprotein is disrupted.

As used herein, the term "vector" refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of preferred vector is an episome, i.e., a nucleic acidcapable of extra-chromosomal replication. Preferred vectors are thosecapable of autonomous replication and/expression of nucleic acids towhich they are linked. Vectors capable of directing the expression ofgenes to which they are operatively linked are referred to herein as"expression vectors". In general, expression vectors of utility inrecombinant DNA techniques are often in the form of "plasmids" whichrefer to circular double stranded DNA loops which, in their vector formare not bound to the chromosome. In the present application, "plasmid"and "vector" are used interchangeably as the plasmid is the mostcommonly used form of vector. However, the invention is intended toinclude such other forms of expression vectors which serve equivalentfunctions and which become known in the art subsequently hereto.

"Transcriptional regulatory sequence" is a generic term used throughoutthe specification to refer to DNA sequences, such as initiation signals,enhancers, and promoters, which induce or control transcription ofprotein coding sequences with which they are operably linked. Inpreferred embodiments, transcription of a recombinant CR gene is underthe control of a promoter sequence (or other transcriptional regulatorysequence) which controls the expression of the recombinant gene in acell-type in which expression is intended. The recombinant gene can alsobe under the control of transcriptional regulatory sequences which arethe same or which are different from those sequences which controltranscription of the naturally-occurring form of the CR proteins.

As used herein, the term "tissue-specific promoter" means a DNA sequencethat serves as a promoter, i.e., regulates expression of a selected DNAsequence operably linked to the promoter, and which effects expressionof the selected DNA sequence in specific cells of a tissue. The termalso covers so-called "leaky" promoters, which regulate expression of aselected DNA primarily in one tissue, but cause expression in othertissues as well.

As used herein, a "transgenic animal" is any animal, preferably anon-human mammal, e.g. a rat, a mouse or pig, in which one or more ofthe cells of the animal includes a transgene. The transgene isintroduced into the cell, directly or indirectly by introduction into aprecursor of the cell, by way of deliberate genetic manipulation, suchas by microinjection or by infection with a recombinant virus. Thelanguage "genetic manipulation" does not include classicalcross-breeding, or in vitro fertilization, but rather is directed to theintroduction of a recombinant DNA molecule. This molecule can beintegrated within a chromosome, or it may be extrachromosomallyreplicating DNA. In the transgenic animals described herein, thetransgene causes cells to express a recombinant form of one or more ofthe subject CR proteins, or alternatively, to disrupt expression of oneor more of the naturally-occurring forms of the CR genes.

As used herein, the term "transgene" refers to a nucleic acid sequencewhich is partly or entirely heterologous, i.e., foreign, to the animalor cell into which it is introduced, or, is homologous to an endogenousgene of the animal or cell into which it is introduced, but which isdesigned to be inserted, or is inserted, into the animal's genome insuch a way as to alter the genome of the cell into which it is inserted(e.g., it is inserted at a location which differs from that of thenatural gene or its insertion results in a knockout). A transgene caninclude one or more transcriptional regulatory sequences and any othernucleic acid, such as introns, that may be necessary for optimalexpression of a selected nucleic acid.

As is well known, genes for a particular polypeptide may exist in singleor multiple copies within the genome of an individual. Such duplicategenes may be identical or may have certain modifications, includingnucleotide substitutions, additions or deletions, which all still codefor polypeptides having substantially the same activity. The term "DNAsequence encoding a CR protein" refers to one or more genes within aparticular individual. Moreover, certain differences in nucleotidesequences may exist between individual organisms, which are calledalleles. Such allelic differences may or may not result in differencesin amino acid sequence of the encoded polypeptide yet still encode aprotein with the same biological activity.

"Cells," "host cells" or "recombinant host cells" are terms usedinterchangeably herein. Such terms refer not only to the particularsubject cell but to the progeny or potential progeny of such a cell.Because certain modifications may occur in succeeding generations due toeither mutation or environmental influences, such progeny may not, infact, be identical to the parent cell, but are still included within thescope of the term as used herein.

A "chimeric protein" or "fusion protein" is a fusion of a first aminoacid sequence encoding one of the subject CR proteins with a secondamino acid sequence defining a domain foreign to and not substantiallyhomologous with any domain of the subject CR protein. A chimeric proteinmay present a foreign domain which is found (albeit in a differentprotein) in an organism which also expresses the first protein, or itmay be an "interspecies", "intergeneric", etc. fusion of proteinstructures expressed by different kinds of organisms.

The language "evolutionarily related to", with respect to nucleic acidsequences encoding CR proteins, refers to nucleic acid sequences whichhave arisen naturally in an organism, including naturally occurringmutants. This language also refers to nucleic acid sequences which,while derived from a naturally occurring CR nucleic, have been alteredby mutagenesis, as for example, combinatorial mutagenesis, yet stillencode polypeptides which have at least one activity of a CR protein.

In one embodiment, the nucleic acid is a cDNA encoding a peptide havingat least one activity of a subject CR proteins. Preferably, the nucleicacid is a cDNA molecule comprising at least a portion of the nucleotidesequence represented in one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, and 13. Apreferred portion of these cDNA molecules includes the coding region ofthe gene.

Preferred nucleic acids encode a CR protein comprising an amino acidsequence at least 60% homologous, more preferably 70% homologous andmost preferably 80%, 90%, or 95% homologous with an amino acid sequenceshown in one of SEQ ID NOs:2, 4, 6, 8, 10, 12, or 14. Nucleic acidswhich encode polypeptides having an activity of a subject CR protein andhaving at least about 90%, more preferably at least about 95%, and mostpreferably at least about 98-99% homology with a sequence shown in oneof SEQ ID NOs:2, 4, 6, 8, 10, 12, or 14 are also within the scope of theinvention. The term "homology" refers to sequence similarity between twopeptides or between two nucleic acid molecules. Homology can bedetermined by comparing a position in each sequence which may be alignedfor purposes of comparison. When a position in the compared sequence isoccupied by the same base or amino acid, then the molecules arehomologous at that position. The degree of homology between sequences isa function of the number of matching or homologous positions shared bythe sequences.

Another aspect of the invention provides a nucleic acid which hybridizesunder high or low stringency conditions to a nucleic acid which encodesa peptide having all or a portion of an amino acid sequence shown in SEQID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12 or SEQ ID NO:14. Appropriate stringency conditions which promoteDNA hybridization, for example, 6.0×sodium chloride/sodium citrate (SSC)at about 45° C., followed by a wash of 2.0×SSC at 50° C., are known tothose skilled in the art or can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Forexample, the salt concentration in the wash step can be selected from alow stringency of about 2.0×SSC at 50° C. to a high stringency of about0.2×SSC at 50° C. In addition, the temperature in the wash step can beincreased from low stringency conditions at room temperature, about 22°C., to high stringency conditions at about 65° C.

Nucleic acids, having a sequence that differs from the nucleotidesequence shown in any of SEQ ID NOs:1, 3, 5, 7, 9, 11, and 13 due todegeneracy in the genetic code are also within the scope of theinvention. Such nucleic acids encode functionally equivalent peptides(i.e., a peptide having a biological activity of a CR protein) butdiffer in sequence from the sequence shown in said sequence listings dueto degeneracy in the genetic code. For example, a number of amino acidsare designated by more than one triplet. Codons that specify the sameamino acid, or synonyms (for example, CAU and CAC each encode histidine)may result in "silent" mutations which do not affect the amino acidsequence of the CR protein. However, it is expected that DNA sequencepolymorphisms that do lead to changes in the amino acid sequences of thesubject CR proteins will exist among vertebrates. One skilled in the artwill appreciate that these variations in one or more nucleotides (up toabout 3-5% of the nucleotides) of the nucleic acids encodingpolypeptides having an activity of an CR protein may exist amongindividuals of a given species due to natural allelic variation. Any andall such nucleotide variations and resulting amino acid polymorphismsare within the scope of this invention.

Fragments of the nucleic acids encoding the active portion of thepresently claimed CR proteins are also within the scope of theinvention. As used herein, a fragment of the nucleic acid encoding theactive portion of a CR protein refers to a nucleic acid having fewernucleotides than the nucleotide sequence encoding the entire amino acidsequence of a CR protein but which nevertheless encodes a peptide havinga biological activity of the CR proteins described herein. Nucleic acidfragments within the scope of the present invention include thosecapable of hybridizing under high or low stringency conditions withnucleic acids from other species for use in screening protocols todetect CR homologs, as well as those capable of hybridizing with nucleicacids from human specimens for use in detecting the presence of anucleic acid encoding one of the subject CR proteins, includingalternate isoforms, e.g. mRNA splicing variants. Nucleic acids withinthe scope of the invention can also contain linker sequences, modifiedrestriction endonuclease sites and other sequences useful for molecularcloning, expression or purification of recombinant forms of the subjectCR proteins.

A nucleic acid encoding a peptide having an activity of an CR proteincan be obtained from mRNA present in any of a number of eukaryoticcells. Nucleic acids encoding CR proteins of the present invention canalso be obtained from genomic DNA obtained from both adults and embryos.For example, a gene encoding a CR protein can be cloned from either acDNA or a genomic library in accordance with protocols herein described,as well as those generally known to persons skilled in the art. A cDNAencoding one of the subject CR proteins can be obtained by isolatingtotal mRNA from a cell, e.g. a mammalian cell, e.g. a human cell,including tumor cells. Double stranded cDNAs can then be prepared fromthe total mRNA, and subsequently inserted into a suitable plasmid orbacteriophage vector using any one of a number of known techniques. Thegene encoding the CR protein can also be cloned using establishedpolymerase chain reaction techniques in accordance with the nucleotidesequence information provided by the invention. The nucleic acid of theinvention can be DNA or RNA. Preferred nucleic acids are the cDNAsrepresented by the sequences shown in SEQ ID NOs:1, 3, 5, 7, 9, 11, and13.

This invention also provides expression vectors containing a nucleicacid encoding a peptide having an activity of an CR protein, operablylinked to at least one transcriptional regulatory sequence. The language"operably linked" refers to linkage of the nucleotide sequence to aregulatory sequence in a manner which allows expression of thenucleotide sequence. Regulatory sequences are art-recognized and areselected to direct expression of the peptide having an activity of a CRprotein. Accordingly, the language "transcriptional regulatory sequence"includes promoters, enhancers and other expression control elements.Such regulatory sequences are described in Goeddel; Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990). The design of the expression vector may depend on such factorsas the choice of the host cell to be transformed and/or the type ofprotein desired to be expressed. In one embodiment, the expressionvector includes a recombinant gene encoding a peptide having an activityof a subject CR protein. Such expression vectors can be used totransfect cells and thereby produce proteins or peptides, includingfusion proteins or peptides, encoded by nucleic acids as describedherein. Moreover, such vectors can be used as a part of a gene therapyprotocol to reconstitute the function of, or alternatively, abrogate thefunction of one of the subject CR proteins in a cell in which a CRprotein is misexpressed.

Another aspect of the present invention concerns recombinant forms ofthe subject CR proteins which are encoded by genes derived fromeukaryotic organisms, e.g. mammals, e.g. humans, and which have at leastone biological activity of a CR protein. The term "recombinant protein"refers to a protein of the present invention which is produced byrecombinant DNA techniques, wherein generally DNA encoding the subjectCR protein is inserted into a suitable expression vector which is inturn used to transform a host cell to produce the heterologous protein.Moreover, the phrase "derived from", with respect to a recombinant geneencoding the recombinant CR protein, includes within the meaning of"recombinant protein" those proteins having an amino acid sequence of anative CR protein of the present invention, or an amino acid sequencesimilar thereto which is generated by mutations including substitutionsand deletions (including truncation) of a naturally occurring CR proteinof a organism. Recombinant proteins preferred by the present invention,in addition to native CR proteins, are at least 60% homologous, morepreferably 70% homologous and most preferably 80% homologous with anamino acid sequence shown in one of SEQ ID NOs:2, 4, 6, 8, 10, 12, or14. Polypeptides having an activity of the subject CR proteins andhaving at least about 90%, more preferably at least about 95%, and mostpreferably at least about 98-99% homology with a sequence of either inSEQ ID NO:2, 4, 6, 8, 10, 12, or 14 are also within the scope of theinvention.

The present invention further pertains to recombinant forms of thesubject CR proteins which are encoded by genes derived from an organismand which have amino acid sequences evolutionarily related to a CRprotein of either SEQ ID NO:2, 4, 6, 8, 10, 12, or 14. The language"evolutionarily related to", with respect to amino acid sequences of thepresent recombinant CR proteins, refers to CR proteins having amino acidsequences which have arisen naturally, as well as mutational variants ofCR proteins which are derived, for example, by combinatorialmutagenesis. Preferred evolutionarily derived CR proteins are at least60% homologous, more preferably 70% homologous and most preferably 80%homologous with an amino acid sequence shown in either SEQ ID NO:2, SEQID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ IDNO:14. Polypeptides having at least about 90%, more preferably at leastabout 95%, and most preferably at least about 98-99% homology with asequence shown in any of SEQ ID NOs:2, 4, 6, 8, 10, 12, or 14 are alsowithin the scope of the invention.

The present invention further pertains to methods of producing thesubject CR proteins. For example, a host cell transfected with a nucleicacid vector directing expression of a nucleotide sequence encoding thesubject CR protein can be cultured under appropriate conditions to allowexpression of the peptide to occur. The peptide can be secreted andisolated from a mixture of cells and medium containing the recombinantCR peptide. Alternatively, the peptide can be retained cytoplasmicallyand the cells harvested, lysed and the protein isolated. A cell cultureincludes host cells, media and other byproducts. Suitable media for cellculture are well known in the art. The recombinant CR peptide can beisolated from cell culture medium, host cells, or both using techniquesknown in the art for purifying proteins including ion-exchangechromatography, gel filtration chromatography, ultrafiltration,electrophoresis, and immunoaffinity purification with antibodiesspecific for such peptide. In a preferred embodiment, the recombinant CRprotein is a fusion protein containing a domain which facilitates itspurification.

This invention also pertains to a host cell transfected to express arecombinant form of at least one of the subject CR proteins. The hostcell can be any prokaryotic or eukaryotic cell. Thus, a nucleotidesequence derived from the cloning of the CR proteins of the presentinvention, encoding all or a selected portion of a protein, can be usedto produce a recombinant form of a CR protein via microbial oreukaryotic cellular processes. Ligating the polynucleotide sequence intoa gene construct, such as an expression vector, and transforming ortransfecting into hosts, either eukaryotic (yeast, avian, insect ormammalian) or prokaryotic (bacterial cells), are standard proceduresused in producing other well-known proteins, e.g. insulin, interferons,human growth hormone, IL-1, IL-2, and the like. Similar procedures, ormodifications thereof, can be employed to prepare recombinant CRproteins, or portions thereof, by microbial means or tissue-culturetechnology in accordance with the subject invention.

A recombinant CR gene can be produced by ligating nucleic acid encodinga subject CR protein, or a portion thereof, into a vector suitable forexpression in either prokaryotic cells, eukaryotic cells, or both.Expression vectors for production of recombinant forms of the subject CRproteins include plasmids and other vectors. For instance, suitablevectors for the expression of a CR protein include plasmids of thetypes: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derivedplasmids, pBTac-derived plasmids and pUC-derived plasmids for expressionin prokaryotic cells, such as E. coli.

A number of vectors exist for the expression of recombinant proteins inyeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 arecloning and expression vehicles useful in the introduction of geneticconstructs into S. cerevisiae (see, for example, Broach et al. (1983) inExperimental Manipulation of Gene Expression, ed. M. Inouye AcademicPress, p. 83, incorporated by reference herein). These vectors canreplicate in E. coli due the presence of the pBR322 ori, and in S.cerevisiae due to the replication determinant of the yeast 2 micronplasmid. In addition, drug resistance markers such as ampicillin can beused.

The preferred mammalian expression vectors contain both prokaryoticsequences to facilitate the propagation of the vector in bacteria, andone or more eukaryotic transcription units that are expressed ineukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo,pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectorsare examples of mammalian expression vectors suitable for transfectionof eukaryotic cells. Some of these vectors are modified with sequencesfrom bacterial plasmids, such as pBR322, to facilitate replication anddrug resistance selection in both prokaryotic and eukaryotic cells.Alternatively, derivatives of viruses such as the bovine papilloma virus(BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can beused for transient expression of proteins in eukaryotic cells. Thevarious methods employed in the preparation of the plasmids andtransformation of host organisms are well known in the art. For othersuitable expression systems for both prokaryotic and eukaryotic cells,as well as general recombinant procedures, see Molecular Cloning ALaboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (ColdSpring Harbor Laboratory Press: 1989) Chapters 16 and 17. In someinstances, the recombinant CR protein can be expressed using abaculovirus expression system. Examples of such baculovirus expressionsystems include pVL-derived vectors (such as pVL1392, pVL1393 andpVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derivedvectors (such as the β-gal containing pBlueBac III).

When expression of a portion of one of the subject CR proteins isdesired, i.e. a truncation mutant, it may be necessary to add a startcodon (ATG) to the oligonucleotide fragment containing the desiredsequence to be expressed. It is well known in the art that a methionineat the N-terminal position can be enzymatically cleaved by the use ofthe enzyme methionine aminopeptidase (MAP). MAP has been cloned from E.coli (Ben-Bassat et al. (1987) J. Bacteriol. 169:751-757) and Salmonellatyphimurium and its in vitro activity has been demonstrated onrecombinant proteins (Miller et al. (1987) PNAS 84:2718-1722).Therefore, removal of an N-terminal methionine, if desired, can beachieved either in vivo by expressing CR-derived polypeptides in a hostwhich produces MAP (e.g., E. coli or CM89 or S. cerevisiae), or in vitroby use of purified MAP (e.g., procedure of Miller et al., supra).

Alternatively, the coding sequences for the polypeptide can beincorporated as a part of a fusion gene including a nucleotide sequenceencoding a different polypeptide. This type of expression system can beuseful under conditions where it is desirable to produce an immunogenicfragment of a CR protein. The nucleic acid sequences corresponding tothe portion of a subject CR protein to which antibodies are to be raisedcan be incorporated into a fusion gene construct which includes codingsequences for a late vaccinia virus structural protein to produce a setof recombinant viruses expressing fusion proteins comprising a portionof the CR protein as part of the virion. It has been demonstrated withthe use of immunogenic fusion proteins utilizing the Hepatitis B surfaceantigen fusion proteins that recombinant Hepatitis B virions can beutilized in this role as well. Similarly, chimeric constructs coding forfusion proteins containing a portion of a CR protein and the polioviruscapsid protein can be created to enhance immunogenicity of the set ofpolypeptide antigens (see, for example, EP Publication No: 0259149; andEvans et al. (1989) Nature 339:385; Huang et al. (1988) J. Virol.62:3855; and Schlienger et al. (1992) J. Virol. 66:2).

The Multiple Antigen Peptide system for peptide-based immunization canalso be utilized to generate an immunogen, wherein a desired portion ofa subject CR protein is obtained directly from organo-chemical synthesisof the peptide onto an oligomeric branching lysine core (see, forexample, Posnett et al. (1988) JBC 263:1719 and Nardelli et al. (1992)J. Immunol. 148:914). Antigenic determinants of the subject CR proteinscan also be expressed and presented by bacterial cells.

In addition to utilizing fusion proteins to enhance immunogenicity,fusion proteins can also facilitate the expression of proteins, such asany one of the CR proteins of the present invention. For example, a CRprotein of the present invention can be generated as aglutathione-S-transferase (GST-fusion protein). Such GST fusion proteinscan enable easy purification of the CR protein, such as by the use ofglutathione-derivatized matrices (see, for example, Current Protocols inMolecular Biology, eds. Ausabel et al. (N.Y.: John Wiley & Sons, 1991)).

Techniques for making fusion genes are known to those skilled in theart. Essentially, the joining of various DNA fragments coding fordifferent polypeptide sequences is performed in accordance withconventional techniques, employing blunt-ended or stagger-ended terminifor ligation, restriction enzyme digestion to provide for appropriatetermini, filling-in of cohesive ends as appropriate, alkalinephosphatase treatment to avoid undesirable joining, and enzymaticligation. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed togenerate a chimeric gene sequence (see, for example, Current Protocolsin Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).

Another aspect of the invention pertains to isolated peptides having anactivity of one of the subject CR proteins. In preferred embodiments, abiological activity of a CR protein includes: promotion of cell cycleprogression (e.g., CR1); ligand-receptor signalling (e.g., CR3);cellular maturation, differentiation, and proliferation (e.g., CR4);enhancement or suppression of DNA replication (e.g., CR5, CR6);promotion of mRNA transcription by stimulating elongation of mRNAtranscription (e.g., CR5, CR6); and transcriptional activation andrepression (e.g., CR8). Other biological activities of the subject CRproteins are described herein or will be reasonably apparent to thoseskilled in the art. A polypeptide having at least one biologicalactivity of the subject CR proteins may differ in amino acid sequencefrom the sequence shown in either SEQ ID NO:2, 4, 6, 8, 10, 12, or 14,but such differences result in a modified polypeptide which functions inthe same or similar manner as the native CR or which has the same orsimilar characteristics of the native CR protein. Various modificationsof a CR protein of the present invention to produce these and otherfunctionally equivalent peptides are described in detail herein. Theterms peptide, protein, and polypeptide are used interchangeably herein.

The present invention also pertains to isolated CR proteins which areisolated from, or otherwise substantially free of other cellularproteins normally associated with the CR protein. The language"substantially free of other cellular proteins" (also referred to hereinas "contaminating proteins") or "substantially pure, substantially purepreparation, or purified preparations" are defined as encompassing CRprotein preparations having less than 20% (by dry weight) contaminatingprotein, and preferably having less than 5% contaminating protein.Functional forms of the subject CR proteins can be prepared, for thefirst time, as purified preparations by using a cloned gene as describedherein. As used herein, the term "purified", when referring to a peptideor DNA or RNA sequence, that the indicated molecule is present in thesubstantial absence of other biological macromolecules, such as otherproteins. The term "purified" as used herein preferably means at least80% by dry weight, more preferably in the range of 95-99% by weight, andmost preferably at least 99.8% by weight, of biological macromoleculesof the same type present (but water, buffers, and other small molecules,especially molecules having a molecular weight of less than 5000, can bepresent). The term "pure" as used herein preferably has the samenumerical limits as the term "purified". "Isolated" and "purified" donot encompass either natural materials in their native state or naturalmaterials that have been separated into components (e.g., in anacrylamide gel) but not obtained either as pure (e.g. lackingcontaminating proteins, or chromatography reagents such as denaturingagents and polymers, e.g. acrylamide or agarose) substances orsolutions.

The term "isolated" as also used herein with respect to nucleic acids,such as DNA or RNA, refers to molecules separated from other DNAs, orRNAs, respectively, that are present in the natural source of themacromolecule. For example, an isolated nucleic acid encoding one of thesubject CR proteins preferably includes no more than 1 0 kilobases (kb)of nucleic acid sequence which naturally immediately flanks thatparticular CR gene in genomic DNA, more preferably no more than 5 kb ofsuch naturally occurring flanking sequences, and most preferably lessthan 1.5 kb of such naturally occurring flanking sequence. The termisolated as used herein also refers to a nucleic acid or peptide that issubstantially free of cellular material, viral material, or culturemedium when produced by recombinant DNA techniques, or chemicalprecursors or other chemicals when chemically synthesized. Moreover, an"isolated nucleic acid" is meant to include nucleic acid fragments whichare not naturally occurring as fragments and would not be found in thenatural state.

Furthermore, isolated peptidyl portions of the subject CR proteins canalso be obtained by screening peptides recombinantly produced from thecorresponding fragment of the nucleic acid encoding such peptides. Inaddition, fragments can be chemically synthesized using techniques knownin the art such as conventional Merrifield solid phase f-Moc or t-Bocchemistry. For example, a CR protein of the present invention can bearbitrarily divided into fragments of desired length with no overlap ofthe fragments, or preferably divided into overlapping fragments of adesired length. The fragments can be produced (recombinantly or bychemical synthesis) and tested to identify those peptidyl fragmentswhich can function as either agonists or antagonists of a CR proteinactivity, such as by microinjection assays.

The structure of the subject CR proteins can be modified for suchpurposes as enhancing therapeutic or prophylactic efficacy, or stability(e.g., ex vivo shelf life and resistance to proteolytic degradation invivo). Such modified peptides, when designed to retain at least oneactivity of the naturally-occurring form of the protein, are consideredfunctional equivalents of the CR proteins described in more detailherein. Such modified peptide can be produced, for instance, by aminoacid substitution, deletion, or addition.

Moreover, it is reasonable to expect that an isolated replacement in CRproteins of the invention of a leucine with an isoleucine or valine, anaspartate with a glutamate, a threonine with a serine, or a similarreplacement of an amino acid with a structurally related amino acid(i.e. conservative mutations) will not have a major effect on thebiological activity of the resulting molecule. Conservative replacementsare those that take place within a family of amino acids that arerelated in their side chains. Genetically encoded amino acids aredivided into four families: (1) acidic=aspartate, glutamate; (2)basic=lysine, arginine, histidine; (3) nonpolar=alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and(4) uncharged polar=glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine aresometimes classified jointly as aromatic amino acids. In similarfashion, the amino acid repertoire can be grouped as (1)acidic=aspartate, glutamate; (2) basic=lysine, arginine histidine, (3)aliphatic=glycine, alanine, valine, leucine, isoleucine, serine,threonine, with serine and threonine optionally be grouped separately asaliphatic-hydroxyl; (4) aromatic=phenylalanine, tyrosine, tryptophan;(5) amide=asparagine, glutamine; and (6) sulfur-containing=cysteine andmethionine. (see, for example, Biochemistry, 2nd ed., Ed. by L. Stryer,W H Freeman and Co.: 1981). Whether a change in the amino acid sequenceof a peptide results in a functional CR homolog can be readilydetermined by assessing the ability of the variant peptide to produce aresponse in cells in a fashion similar to the wild-type CR protein orpeptide. Peptides in which more than one replacement has taken place canreadily be tested in the same manner.

Another aspect of the invention pertains to an antibody or antibodypreparation specifically reactive with at least one epitope of at leastone of the subject CR proteins. For example, by using immunogens derivedfrom the present CR proteins, based on the cDNA sequences,anti-protein/anti-peptide antisera or monoclonal antibodies can be madeby standard protocols (See, for example, Antibodies: A Laboratory Manualed. by Harlow and Lane (Cold Spring Harbor Press: 1988)). A mammal suchas a mouse, a hamster or rabbit can be immunized with an immunogenicform of the peptide (e.g., CR protein or an antigenic fragment which iscapable of eliciting an antibody response). Techniques for conferringimmunogenicity on a protein or peptide include conjugation to carriersor other techniques well known in the art. An immunogenic portion of thesubject CR proteins can be administered in the presence of adjuvant. Theprogress of immunization can be monitored by detection of antibodytiters in plasma or serum. Standard ELISA or other immunoassays can beused with the immunogen as antigen to assess the levels of antibodies.In a preferred embodiment, the subject antibodies are immunospecific forantigenic determinants of the CR proteins of the present invention, e.g.antigenic determinants of a protein represented by one of SEQ ID NOs:2,4, 6, 8, 10, 12, or 14 or a closely related human or non-human mammalianhomolog (e.g. 90 percent homologous, more preferably at least 95 percenthomologous). In yet a further preferred embodiment of the presentinvention, the anti-CR protein antibodies do not substantially crossreact (i.e. react specifically) with a protein which is: e.g. less than90 percent homologous to one of SEQ ID NOs:2, 4, 6, 8, 10, 12, or 14;e.g. less than 95 percent homologous with one of SEQ ID NOs:2, 4, 6, 8,10, 12, or 14; e.g. less than 98-99 percent homologous with one of SEQID NOs:2, 4, 6, 8, 10, 12, or 14. The language "not substantially crossreact" means that the antibody has a binding affinity for anon-homologous protein which is less than 10 percent, more preferablyless than 5 percent, and even more preferably less than 1 percent, ofthe binding affinity for a protein of SEQ ID NOs:2, 4, 6, 8, 10, 12, or14.

Following immunization, anti-CR antisera can be obtained and, ifdesired, polyclonal anti-CR antibodies isolated from the serum. Toproduce monoclonal antibodies, antibody producing cells (lymphocytes)can be harvested from an immunized animal and fused by standard somaticcell fusion procedures with immortalizing cells such as myeloma cells toyield hybridoma cells. Such techniques are well known in the art, aninclude, for example, the hybridoma technique (originally developed byKohler and Milstein, (1975) Nature, 256: 495-497), the human B cellhybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc. pp. 77-96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with a CR protein of thepresent invention and monoclonal antibodies isolated from a culturecomprising such hybridoma cells.

The term "antibody" as used herein is intended to include fragmentsthereof which are also specifically reactive with one of the subject CRproteins. Antibodies can be fragmented using conventional techniques andthe fragments screened for utility in the same manner as described abovefor whole antibodies. For example, F(ab')₂ fragments can be generated bytreating antibody with pepsin. The resulting F(ab')₂ fragment can betreated to reduce disulfide bridges to produce Fab' fragments.Antibodies of the present invention are further intended to includebispecific and chimeric molecules having an anti-CR portion.

Both monoclonal and polyclonal antibodies (Ab) directed against CR or CRvariants, and antibody fragments such as Fab' and F(ab')₂, can be usedto block the action of a CR proteins and allow the study of the role ofthe particular CR protein of the present invention in cell signalling.

The nucleotide sequence determined from the cloning of the subject CRproteins from a human cell line allows for the generation of probesdesigned for use in identifying CR homologs in other human cell types,as well as CR homologs from other animals. For instance, the presentinvention also provides a probe/primer comprising a substantiallypurified oligonucleotide, wherein the oligonucleotide comprises a regionof nucleotide sequence which hybridizes under stringent conditions to atleast 10 consecutive nucleotides of sense or anti-sense sequence of oneof SEQ ID NOs: 1, 3, 5, 7, 9, 11, or 13, or naturally occurring mutantsthereof. In preferred embodiments, the probe/primer further comprises alabel group attached thereto and is able to be detected, e.g. the labelgroup is selected from the group consisting of radioisotopes,fluorescent compounds, enzymes, and enzyme co-factors. Such probes canbe used as a part of a test kit for measuring a level of an CR nucleicacid in a sample of cells from a patient; e.g. measuring a CR mRNAlevel; e.g. determining whether a genomic CR gene has been mutated ordeleted.

In addition, nucleotide probes can be generated from the cloned sequenceof the subject CR proteins, which allow for histological screening ofintact tissue and tissue samples for the presence of a CR mRNA. Use ofprobes directed to CR mRNAs, or to genomic CR sequences, can be used forboth predictive and therapeutic evaluation of allelic mutations whichmight be manifest in a variety of disorders including cancer,immunodeficiencies, autoimmune disorders, developmental abnormalities,infectious diseases, toxic damage due to irradiation, chemicals, andother noxious compounds or natural products. Used in conjunction withanti-CR antibody immunoassays, the nucleotide probes can help facilitatethe determination of the molecular basis for a developmental disorderwhich may involve some abnormality associated with expression (or lackthereof) of a CR protein. For instance, variation in CR synthesis can bedifferentiated from a mutation in the CR coding sequence.

Also, the use of anti-sense techniques (e.g. microinjection of antisensemolecules, or transfection with plasmids whose transcripts areanti-sense with regard to a CR mRNA or gene sequence) can be used toinvestigate the normal cellular function of each of the novel CRproteins, e.g. in cell signalling. Such techniques can be utilized incell culture, but can also be used in the creation of transgenicanimals.

Furthermore, by making available purified and recombinant CR proteins,the present invention facilitates the development of assays which can beused to screen for drugs which are either agonists or antagonists of thenormal cellular function of the subject CR proteins, or of their role incell signalling.

In another aspect, the invention features transgenic non-human animalswhich express a recombinant CR gene of the present invention, or whichhave had one or more of the subject CR gene(s), e.g. heterozygous orhomozygous, disrupted in at least one of the tissue or cell-types of theanimal.

In another aspect, the invention features an animal model for disordersrelated to cell signalling, which has a CR allele which ismis-expressed. For example, a mouse can be bred which has a CR alleledeleted, or in which all or part of one or more CR exons are deleted.Such a mouse model can then be used to study disorders arising frommis-expressed CR genes.

This invention is further illustrated by the following examples which inno way should be construed as being further limiting. The contents ofall cited references (including literature references, issued patents,published patent applications, and co-pending patent applications) citedthroughout this application are hereby expressly incorporated byreference.

EXAMPLES Example I Construction of cDNA Library Containing Clones ofLigand-Induced Genes

Human peripheral blood mononuclear cells (PBMCs) were isolated byFicoll/Hypaque discontinuous centrifugation, and cultured at 10⁶cells/ml in complete medium comprised of RPMI 1640 (GIBCO Laboratories,Grand Island, N.Y.) supplemented with 10% heat-inactivated (56° C., 30min) calf serum (Sterile Systems, Inc., Logan, Utah), 50 mg/mlL-glutamine, and 50 units/ml penicillin. T-cells were activated bystimulation of the CD3 component of the T-cell receptor complex with ananti-CD3 reactive monoclonal antibody (OKT3, 1:10,000 dilution, OrthoPharmaceuticals, Raritan, N.J.) in the presence of absence of 10 mg/mlCHX, and DNA synthesis was monitored at 48-52 hr by adding 0.5 mCi ³H!-thymidine to 200 ml aliquots of cell cultures in 96-well microtiterplates. Cultures were harvested onto glass fiber filters, radioactivitywas counted by liquid scintillation, and ³ H!-thymidine incorporationwas calculated as cpm/10⁴ cells/hr.

IL-2R-positive T-cell blasts were prepared by stimulation of PBMCs withOKT3 for 3 days, after which the cells were washed and replaced inculture for an additional 11 days in the presence of 500 pM IL-2. Thecells were subsequently washed and placed in culture in the absence ofIL-2 for 36 hr, followed by a 12 hr stimulation with 50 ng/ml phorbol12, 13 dibutyrate (PdBu) to augment high-affinity IL-2R expression.Cells were washed free of PdBU and placed in culture for 12 hr prior torestimulation. Such treatment enabled the generation of a G₀ /G₁-synchronized cell population, made up of greater than 90% T8-positive Tlymphocytes (Gullberg et al. (1986) J. Exp. Med. 163:270-284).

Human IL-2R-positive T-cell blasts were cultured in the presence of 1 nMIL-2, 10 mg/ml CHX, 250 mM 4-thiouridine (Sigma Chemical Co., St. Louis,Mo.) and 2.5 mCi/ml 5,6-³ H!-uridine (48 Ci/mmole, Amersham, ArlingtonHeights, Ill.) for 2 hr. CHX was included in the 2 hr IL-2 stimulationof the IL-2R-positive, G₀ /G₁ -synchronized human T-cells from which thecDNA library was generated in order to isolate immediate-early genes,and also to possibly superinduce the expression of low-abundancemessages. Total RNA was isolated essentially as described by Caligiuriet al. ((1989) J. Exp. Med. 171:1509-1526), and the4-thiouridine-labelled RNA purified by passage over a phenylmercuryagarose column as described by Woodford et al. ((1988) Anal. Biochem.171:166-172). The cells were labelled with 4-thiouridine duringstimulation, to enable isolation of only those transcripts which weresynthesized during the period of IL-2 and CHX treatment (Stetler et al.(1984) Proc. Nat. Acad. Sci. (USA) 81:1144-1148) and Woodford et al.((1988) Anal. Biochem. 171:166-172). Fractionation of total cellular RNAresulted in a 10-fold enrichment for newly-synthesized transcripts.

This thiol-selected RNA was used in the synthesis of Not-1primer/adapter-primed cDNA, utilizing the Riboclone cDNA SynthesisSystem (Promega, Madison, Wis.) according to manufacturers instructions.After addition of EcoRI adapters (Promega), Not-1 digestion, and sizeselection for fragments greater than 500 base pairs (bp), the cDNA wasligated directionally into an EcoRI- and Not-1-digested pBluescript IISK+ plasmid vector (Stratagene, La Jolla, Calif.), followed bytransformation into Epicurian Coli XL-1 Blue competent cells(Stratagene). A cDNA library of approximately 10,000 clones resulted.

Example II Screening of cDNA Library for Clones ContainingLigand-Induced Genes

About 10% of the cDNA library was then screened using radiolabelled cDNAprobes made from mRNA isolated from T-cells induced with IL-2 or fromuninduced cells as follows. Single-stranded ³² P!-labelled cDNA probeswere prepared from poly(A)⁺ RNA isolated from human T-cell blastsstimulated for 2 hr with medium (unstimulated probe), or 1 nM IL-2 and10 mg/ml CHX (stimulated probe). Total cellular RNA was prepared asdescribed by Caligiuri et al. ((1989) J. Exp. Med. 171:1509-1526), andpoly(A)⁺ RNA was isolated by three passages over an oligo-dT-cellulosecolumn (5 Prime-3 Prime, West Chester, Pa.). First strand cDNA synthesiswas performed with an oligo-dT 12-18 primer (United States BiochemicalCorp., Cleveland, Ohio), using the Riboclone cDNA Synthesis System(Promega, Madison, Wis.) according to manufacturers instructions, withthe exception of dCTP at a final concentration of 35 mM and the additionof 2.5 mCi/ml ³² P!-dCTP. Hybridization was carried out for 72-96 hr at42° C. in 50% formamide, with a final probe concentration ofapproximately 2×10⁶ cpm/ml (W. M. Strauss, in Current Protocols inMolecular Biology, (1989) pp. 6.3.1-6.3.6). Subsequent to hybridization,filters were washed repeatedly at 62° C. in 0.1×SSC (1×SSC=0.15M NaCl,0.015M sodium citrate, pH 7.0), 0.1% SDS and placed on film (KodakXAR-5) with Dupont Cronex intensifying screens overnight at -70° C. Theinitial screening yielded 18 putative positive clones which exhibiteddifferential hybridization to the stimulated and unstimulated probesafter three independent screens. These clones were isolated for furthercharacterization by Northern Blot analysis.

Total cellular RNA was isolated by the guanidine thiocyanate methoddescribed by Caligiuri et al. (ibid.), and denatured in glyoxal andDMSO. The RNA was fractionated on a 1% agarose gel in 0.01M NaH₂ PO₄with 0.5 mg/ml ethidium bromide (Selden, Current Protocols in MolecularBiology, (1989) pp. 4.9.5-4.9.8). To estimate sizes of RNA transcripts,a 0.24-9.5 kb RNA ladder (Bethesda Research Laboratories, Gaithersburg,Md.) was run alongside the cellular RNA samples. After visualizationunder ultraviolet light, the RNA was transferred to nitrocellulose bycapillary transfer in 10×SSC. Plasmids were purified from the clones ofinterest, and the Not-1- and EcoRI-excised inserts ³² P!-labelled withrandom primers. Hybridization was carried out in 50% formamide at 42° C.for 48-72 hr, followed by repeated washes in 0.1×SSC, 0.1% SDS at56°-62° C. (Selden, ibid.). Filters were exposed to Kodak XAR-5 filmwith Dupont Cronex intensifying screens, and specific bands quantitatedwith an EC densitometer (EC Apparatus Corp., St. Petersburg, Fla.).

In as much as CHX was included in both the library and probepreparation, it was essential to verify that the differential expressionof putative clones observed upon colony screening was not due solely tothe effects of this drug. In addition, determination of the sizes andpatterns of induction of the RNA transcripts was necessary to enableestimation of the redundancy of the clones. Therefore, Northern blotanalysis was performed with RNA isolated from human IL-2R-positiveT-cell blasts stimulated with either CHX or IL-2 alone, or with acombination of the two agents.

Hybridization of the RNA with probes generated from the inserts of eachof the 18 putative clones resulted in the identification of 4 clonesthat were solely CHX-induced. For the remaining 14 clones, the inductionby the combination of IL-2 and CHX could not be accounted for by theeffects of CHX alone. Based upon the patterns of induction andapproximate sizes of the RNA transcripts, 8 readily distinguishable andapparently unique IL-2-induced genes were discerned, as partialsequences, among these 14. These are described in Table X.

                  TABLE X    ______________________________________    Some Characteristics of the Eight Proteins Cloned                          Insert   RNA                          (kb)     (bases)                          Size of  Size of                          Partial  Partial                                          IL-2    Clone Nucleotide Sequence                          Sequence Sequence                                          Induction    ______________________________________    CR1   nucleotide 857 to 2406 of                          1.6      2406   24    (1A8) SEQ ID NO:1    CR2   nucleotides 1 to 163 and                          1.1      1283   7    (1F5) nucleotides 1093 to 1283          of SEQ ID NO:3    CR3   nucleotides 718 to 901                          2.0      2450   22    (10A8)          and nucleotides 2265 to          2450 of SEQ ID NO:5    CR4   nucleotides 2101 to 2291                          1.0      2946   6    (10D6)          and nucleotides 2679 to          2928 of SEQ ID NO:7    CR5   nucleotides 763 to 902                          1.4      2020   >50    (10F9)          and nucleotides 1641 to          2020 of SEQ ID NO:9    CR6   nucleotides 310 to 513                          1.0      1066   5    (11B2)          and nucleotides 687 to          1066 of SEQ ID NO:11    CR7   corresponds to  0.7      2400   17    (11E6)          nucleotides of pim-1          sequence in Selten et al.    CR8   nucleotides 1721 to 1915                          1.5      2980   7    (13E2)          of SEQ ID NO:13    ______________________________________

The original designations of the CR clones are included in parenthesesin the left-hand column of Table II. The original designations are usedherein to refer to the partial sequences shown in the column second fromthe left in Table II. As shown in Table II and in FIGS. 8A-8H, three ofthe genes, CR1, CR3, and CR5, were induced by IL-2 alone, while five ofthe genes, CR2, CR4, CR6, CR7, and CR8, were induced by both CHX andIL-2. In several instances, the combination of IL-2 and CHX resulted ina marked synergistic induction.

Example III Kinetic Analysis of IL-2-Induced Gene Expression

The temporal expression of the novel, IL-2-induced genes was determinedby Northern blot analysis, using RNA isolated from human IL-2R-positiveT-cell blasts after IL-2 stimulation in the presence or absence of CHX.Northern blots were prepared with 15 mg total RNA isolated from G₀ /G₁-synchronized human T-cells stimulated for 0, 0.5, 1, 2, 4, or 8 hourswith 1 nM IL-2 or IL-2+10 mg/ml CHX. Filters were probed with the cDNAinserts of the IL-2-induced clones.

As shown in FIGS. 9A-9H, two of the genes, 1A8 (FIG. 9A) and 10D6 (FIG.9B), exhibited rapid induction, reaching peak levels within 1-4 hr ofIL-2 stimulation and returning to basal levels after 8 hr, while theother six clones (FIGS. 9C-9H) remained at elevated levels for at least8 hr after IL-2 treatment. The magnitude of IL-2 induction of steadystate RNA levels of the clones ranged from an approximately 5-foldelevation of clone 11B2 (FIG. 9F) to a greater than 50-fold stimulationof clone 10F9 (FIG. 9E) during the interval examined. These results arealso summarized in Table II. Several of the clones were superinduced byCHX, with an increase observed in both the magnitude and duration of theIL-2 response.

The kinetics of induction of previously characterized IL-2-responsivegenes have been found to range from those such as c-fos, which arerapidly and transiently induced within minutes of IL-2 stimulation(Dautry et al. (1988) J. Biol. Chem. 263:17615-17620), to those whichremain at elevated levels through G₁ to S phase entry (Sabath et al.(1990) J. Biol. Chem. 265:12671-12678).

Example IV Sequence Analysis of Clones Containing Ligand-Induced Genes

To verify the redundancy of the clones as estimated from Northernanalysis, as well as to determine the identities of the genes, the cDNAclones were subjected to sequence analysis.

Plasmids were isolated from the clones of interest essentially asdescribed by Kraft et al. ((1988) Biotechniques 6:544-547), and vectorprimers were used to sequence the termini of the cDNA inserts, employingthe Sequenase 2.0 dideoxy sequencing kit (United States Biochemical,Cleveland, Ohio). Approximately 200 bases of sequence were attained fromeach end of the inserts. These partial sequences are described in TableII. Searches of the GenBank and EMBL data bases were performed with theFASTA program as described by Pearson et al. ((1988) Proc. Natl. Acad.Sci. (USA) 85:2444-2448).

The combination of sequence and Northern analyses revealed that the 14putative IL-2-induced clones consisted of 8 unique genes, three ofwhich, 1A8, 11B2, and 13E2, were isolated three times each. Searches ofthe GenBank and EMBL data bases with the partial sequences enabled theidentification of one clone, 11E6, as pim-1, a previously characterizedIL-2-induced gene (Dautry et al. (1988) J. Biol. Chem. 263:17615-17620;and Kakut-Houri et al. (1987) Gene 54:105-111) which encodes a 33 kDcytoplasmic kinase (Telerman et al. (1988) Mol. Cell. Biol.8:1498-1503).

Thus, by utilizing the method of the invention seven unique IL-2 inducedgenes were cloned, representing novel human genes. These clones wereidentified after screening only approximately 800 library colonies, andthus, it is estimated that as many as 80 additional novel IL-2-inducedgenes remain to be detected in the 10,000-clone library.

To determine the complete sequences of these clones described in TableII the original partial cDNAs were used as probes to screen a secondcDNA library. It is standard procedure to use partial cDNA insertsidentified by an initial screen of a cDNA library to make radiolabeledcDNA probes to screen a second library to obtain clones with theportions missing in the initial cDNA clones. This was done, briefly, asfollows: a second cDNA library was prepared from mRNA obtained fromhuman T cells stimulated for two hours with interleukin-2 in thepresence of cycloheximide by cloning into the λgt-10 phage vector usingstandard methods. (Sambrook, J. et al. Molecular Cloning: A LaboratoryManual (Cold Spring Harbor Laboratory Press, New York, 1989) pp.2.82-2.122).

This second cDNA library was then screened using as probes each of thecDNA fragments obtained from the first, thiol-selected cDNA library.Candidate clones that corresponded to the correct size according to themRNA were then subcloned and sequenced. The complete cDNA sequences (andthe predicted amino acid sequences) of seven out of eight of theseclones are set forth in SEQ ID NOs:1-14 and FIGS. 1-7. The complete cDNAsequence (and the predicted amino acid sequence) of the eighth clone wasdetermined to be identical to that of the IL-2 induced gene pim-1. Thenucleotide sequence as well as the predicted amino acid sequence ofpim-1 are set forth at page 605 in Selten, G. et al. (1986) Cell46:603-611.

Example V Determination of Sensitivity of IL-2-Induced Gene Expression

As a further means of characterizing the regulation of expression ofthese genes, the sensitivity of induction to the known IL-2 functionalantagonist was investigated. Human IL-2R-positive T-cell blasts werestimulated with IL-2 in the absence or presence of 0.5 mMdibutyryl-cAMP, a concentration of the membrane-permeant cAMP analogsufficient to inhibit IL-2-mediated G₁ progression without adverselyaffecting cellular viability. The effect of an equivalent molar amountof sodium butyrate, which does not inhibit the IL-2 response, was alsotested to control for the actions of free butyric acid.

Northern blots were prepared as follows: Human IL-2R-positive T cellswere treated with 1 nM IL-2 alone or in combination with 0.5 mMdibutyryl cAMP or sodium butyrate (NaBt) for 1, 2, or 4 hours. Filterswere prepared with 15 mg total RNA and hybridized with cDNA inserts orthe IL-2 induced clones.

These analyses demonstrate that the IL-2 induction of one gene, 1A8(FIG. 10A) is markedly inhibited when the intracellular level of cAMP israised by the addition of dibutyryl cAMP, whereas the expression of twoothers, 10D6 (FIG. 10B) and 13E2 (FIG. 10C), is augmented approximately3-fold. By comparison, the expression of five of the genes was notaffected by elevated cAMP (FIGS. 10D-10H). Thus, the sequences in clone1A8 may be involved in T-cell proliferation. The fact that not all geneswere sensitive to cAMP indicated that the observed results were not dueto non-specific effects, and furthermore that the previously documenteddown-regulation of IL-2R binding capacity by cAMP (Johnson et al. (1990)J. Immunol. 145:1144-1151) could not account for the inhibition of geneexpression.

Example VI Determination of Role of T-cell Receptor Activation in theStimulation of Expression of IL-2-Induced Genes

In order to determine if activation of the T-cell receptor mediates thestimulation of expression of cytokine IL-2-induced genes, the followingstudy was performed. Northern blots were prepared from 20 mg totalcellular RNA isolated from human peripheral blood mononuclear cells(PBMCs) stimulated with a monoclonal antibody (OKT3) specific to the CD3component of the T-cell antigen receptor complex. Blots were probed withcDNA inserts of the IL-2-induced clones. Data was determined as themean±SEM (n=6).

By isolation of RNA at early time intervals, it was possible to identifythose genes which were induced by T-cell receptor triggering in theabsence of IL-2 effects. As shown in FIGS. 11A-11H, only one of thegenes, 10D6 exhibited heightened levels of expression after 2 hr ofT-cell receptor activation, while the seven others were apparentlyinsensitive to this stimulus. Two of the clones, 1F5 and 11B2, wereundetectable, even after seven days of autoradiographic exposure of theNorthern blots. Two other genes, 11E6 and 13E2, were expressed atrelatively high levels regardless of the stimulus; activation withanti-CD3 did not induce RNA expression beyond the level observed byculture in medium alone. Identical results were obtained after 1 and 4hr of stimulation.

To determine whether the cells were actually activated via CD3, aliquotsof the cells were left in culture for 52 hr in the presence of 10 mg/mlCHX, alone, OKT3 alone, or OKT3+CHX, after which cell cycle progressionwas monitored by ³ H!-thymidine incorporation into RNA.

As shown in FIG. 12, the cells were sufficiently stimulated by anti-CD3.Thus, the T-cell receptor-induced expression of only one of the geneswas comparable to that seen with IL-2 stimulation, while the expressionof the seven others was unique to the IL-2 signaling pathway. Thus, themethods described herein to identify IL-2-induced gene successfullyselected and enriched for these genes that are highly specific forcytokine (IL-2) activation.

Of the 8 IL-2 induced G₁ progression genes reported here, only oneappears to also be induced during the T cell receptor-mediatedcompetence phase of the cell cycle. Thus, while several genes such asc-fos, c-myc and c-raf-1 are known to be induced during both the initialG₀ -G₁ and subsequent G₁ -S phase transitions, the expression of anumber of IL-2-stimulated genes is unique to the latter event. Inaddition, the immediate-early genes reported here appear to define aclass distinct from the IL-2-induced genes isolated by Sabath et al.((1990) J. Biol. Chem. 265:12671-12678). These investigators utilized adifferential screening procedure to isolate genes expressed at the G₁ /Sphase boundary in a murine T helper clone which was stimulated with IL-2for 20 hr in the absence of protein synthesis inhibitors. In this case,the expression of only 3 of the 21 clones isolated was inhibited by CHX,while the remainder were insensitive to this agent. This pattern ofregulation markedly contrasts with the CHX superinduction observed withthe immediate-early IL-2-induced genes described here. Moreover, theseobservations indicate that IL-2 stimulates a complex program of geneexpression, ranging from those genes induced very early in G₁ throughthose subsequently expressed at the G₁ /S phase transition.

Example VII Cloning and Analysis of CR8

As described above, the CR8 gene encodes a novel basic helix-loop-helix(bHLH) protein. While the CR8 transcript is ubiquitously expressed inmany tissues, it is induced by IL-2 as well as by IL-3 incytokine-dependent lymphoid cell lines. In an IL-2-dependent human Tcell line Kit 225, the CR8 transcript is induced not only by IL-2, butalso by interferon b and forskolin, which elevates intracellular cAMP.The bHLH domain of CR8 shows the highest structural homology to aDrosophila transcriptional repressor hairy. The recombinant CR8 proteinbinds preferentially to the Class B E-box DNA sequence (CACGTG), whichis found in the promoter/enhancer regions of a number of genesassociated with cell growth and differentiation, suggesting that CR8 mayregulate the transcription of such genes.

The cloning of the full-length cDNA for CR8 is described in detailherein. The predicted amino acid sequence revealed that CR8 contains ahelix-loop-helix (HLH) domain, characteristic for transcription factors.The HLH domain is a dimerization motif characterized by the twoamphipathic α-helices separated by a nonhelical loop of variable length(Davis, R. L. et al.(1990) Cell 60:733-746). Most of the HLH familymembers possess a cluster of basic amino acid residues immediatelyN-terminal to the HLH region basic helix-loop-helix (bHLH)!, which arerequired for site-specific DNA binding, while others lack the basicregion and function as negative regulators of DNA binding (Benezra, R.et al. (1990) Cell 61:49-59; Ellis, H. M. et al. (1990) Cell 61:27-38;Garrell, J. et al. (1990) Cell 61:39-48.22,28). A wide variety ofdevelopmental processes are regulated by HLH proteins; the MyoD familyof myogenic transcription factors directly induce the expression ofmuscle-specific genes, thereby functioning as master regulators ofmuscle cell lineage specification (reviewed in (Edmonson, D. G. et al.(1993) J. Biol. Chem. 268:755-758; Weintraub, H. (1993) Cell75:1241-1244)). The crucial role of the bHLH protein encoded by thetal-l/SCL gene in hematopoiesis, originally discovered as a chromosomalbreakpoint in leukemia (Begley, C. G. et al. (1989) Proc. Natl. AcadSci. USA 86:10128-10132; Chen, G. et al. (1990) EMBO J. 9:415-424;Finger, L. et al. (1989) Proc. Natl. Acad. Sci. USA 86:5039-5043), isillustrated by the absence of blood formation in tal-l null mutant mice(Shivdasani, R. A. et al. (1995) Nature 373:432-434).

The regulation of immunoglobulin (Ig) gene expression has beenextensively studied, and has been shown to be controlled by numeroustranscription factors that recognize specific DNA sequences in the Igenhancers (Kadesch, T. (1992) Immunol. Today 13:31-36). Recent reportson E2A null mutant mice that lack mature B cells clearly depict theimpact of these bHLH proteins on B cell development (Bain, G. et al.(1994) Cell 79:885-892; Zhuang, Y. et al. (1994) Cell 79:875-884).Genetic analysis of neural cell fate and sex determination in Drosophilaprovided in vivo evidence for interaction between bHLH proteins(reviewed in (Jan, Y. N. et al. (1993) Cell 75:827-830)). For instance,bHLH proteins encoded by daughterless (da) and the achaete-scute complex(AS-C) heterodimerize and positively regulate sensory organ formation.On the other hand, the genes encoding negative regulators such as hairyand extramacrochaetae are required to control the appropriate pattern ofneural precursor distribution. Moreover, because cell differentiation isoften associated with the suppression of proliferation, some HLHproteins have also been implicated in the regulation of cell growth. Oneof the most extensively studied may be Myc, a bHLH protein encoded bythe c-myc oncogene (reviewed in Marcu, K. B. et al. (1992) Annu. Rev.Biochem. 61:809-860). The negative regulator Id proteins which inhibitdifferentiation by forming inactive heterodimers with bHLH proteins,thereby may be required for proliferation. For example, the level of Idexpression is higher in undifferentiated proliferating cells (Benezra,R. et al. (1990) Cell 61:49-59). Also, antisense oligonucleotide againstId mRNA inhibits re-entry to the cell cycle (Barone, M. V. et al. (1994)Proc. Natl. Acad. Sci. USA 91:4985-4988; Hara, E. et al. (1994) J. Biol.Chem. 269:2139-2145), and cell cycle progression is accelerated in Id2stable transfectant cell lines (Ivarone, A. et al. (1994) Genes & Dev8:1270-1284).

The following Materials and Methods were used in this Example:

Cell Culture and Reagents:

Human T cells were prepared as described previously (Beadling, C. et al.(1993) Proc. Natl. Acad. Sci. USA 90:2719-2723); in short, peripheralblood mononuclear cells were cultured in RPMI 1640 supplemented with 10%(v/v) heat-inactivated fetal calf serum (FCS) and antibiotics in thepresence of OKT3 (Ortho Pharmaceuticals) for 3 days, then for anadditional 11 days in the presence of IL-2 (Takeda Chemical). The cellswere subsequently removed from of IL-2 for 36 hr, followed by a 12 hrstimulation with phorbol-12, 13-dibutyrate (Sigma) to augment theexpression of high-affinity IL-2 receptor. Such treatment enabled thegeneration of a G0/G1-synchronized cell population, comprised of >90%CD8+ T lymphocytes (Gullberg, M. et al. (1986) J. Exp. Med.163:270-284). Kit 225 is an IL-2-dependent human T cell line (Hori, T.et al. (1987) Blood 70:1069-1072). Ba/F3 and CTLL2 are mouse cell linesdependent on IL-3 and IL-2, respectively. Both Kit 225 and CTLL2 weremaintained in RPMI1640 supplemented with 10% (v/v) FCS and 500 pMrecombinant human IL-2. Ba/F3 was maintained in RPMI1640 supplementedwith 10% (v/v) FCS and 5% (v/v) conditioned medium from fibroblaststransfected with mouse IL-3 as a source of IL-3. Recombinant mouse IL-3was purchased from Genzyme. Before using for experiments, cell lineswere made quiescent by growth factor deprivation for 72 hr for Kit 225,12 hr for Ba/F3 and 2 hr for CTLL2.

Forskolin was obtained from Sigma. Human interferon (IFN) β was fromGIBCO BRL. Proliferation was monitored by measuring the incorporation of³ H-methyl!thymidine (Amersham) into ten thousand cells incubated withindicated reagents in 200 fl for 24 hr at 37° C. The culture was pulsedwith 0.5 fCi ³ H!thymidine for the last 4 hr prior to harvest.

Northern Hybridization:

Total cellular RNA was isolated by RNAzolB (Tel-Test) and fractionatedon a 1.2% agarose formaldehyde gel. RNA was visualized with ethidiumbromide. After electrophoresis, RNA was transferred and fixed toHybond-N+ nylon membrane (Amersham) with 40 mM NaOH. Multiple TissueNorthern Blot membranes were purchased from Clontech. Membranes werehybridized with the radiolabeled probe for 3 hr to overnight at 65° C.in Rapid-Hyb hybridization solution (Amersham), washed twice with2×SSC/0.1% SDS (1×SSC=150 mM NaCl/15 mM sodium citrate, pH 7.0) at roomtemperature for 15 min, once with 0.5×SSC/0.1% SDS at 60° C. for 15 minand subjected to autoradiography.

cDNA Library Screening:

The λgt10 cDNA libraries were constructed and screened according to thestandard molecular biology procedure (Sambrook, J., E. F. Fritsch, andT. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, SecondEdition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).Poly(A)+ RNA was isolated from IL-2-stimulated normal human T cellsprepared as above and cDNA synthesis was primed with both oligo(dT)12-18and random hexamers. The recombinant phages were screened withradiolabeled CR8 insert. For mouse CR8, aλgt10 cDNA library fromIL-2-stimulated mouse splenocytes were screened with human CR8 insertunder low-stringency condition.

Sequence Analysis:

CR8 cDNA sequence was analyzed by the fluorescence-baseddideoxynucleotide termination method (Taq DyeDeoxy™ Terminator CycleSequencing Kit, Perkin Elmer) on the Applied Biosystems Model 373A DNAsequencer. Consensus sequences were constructed and analyzed with thehelp of the University of Wisconsin GCG software package. The BLASTalgorithm from the National Center for Biotechnology Information (NCBI)was also employed for nucleotide and amino acid sequence homology search(Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403-410).

Preparation of Recombinant Proteins:

The recombinant CR8 protein with histidine-tag was prepared using theXpress™ System (Invitrogen) according to the manufacturer's protocol.cDNA corresponding to the CR8 bHLH domain was obtained by PCR. Thesequences of the primers, 5'-GGGGTCTACCAGGGATGTAC-3' (SEQ ID NO:15) forthe 5' side, and 5'-GTAAACCACTCTGCAGGGCAATGA-3' (SEQ ID NO:16) for the3' side, were slightly different from the final consensus sequence forCR8, but the difference did not affect the core bHLH motif. The PCRproduct was cloned into pT7Blue T-vector (Novagen) and subsequently intopRSET-A vector at BamHI and HindIII sites. Constructs were confirmed byDNA sequencing. The protein was overexpressed in JM109 at 37° C. in thepresence of isopropylthio-b-D-galactoside (IPTG) by infecting thebacteria with M13 phages that contain the T7 RNA polymerase gene. Thecells were lysed with 100 fg/ml lysozyme in native binding buffer (20 mMsodium phosphate, pH 7.8, 500 mM NaCl), the lysate was loaded on aProBond™ Ni2+ column, and the recombinant protein was eluted withnative-imidazole elution buffer (20 mM sodium phosphate, pH 6.0, 500 mMNaCl, 500 mM imidazole). The protein was then dialyzed against lysisbuffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol,0.1% Triton X-100, 1 mM DTT) and concentrated on a Microcon 10 (Amicon).To confirm the purity and the quantity, the protein was fractionated ona 12% SDS-polyacrylamide gel with protein standards of knownconcentrations and visualized by silver staining. The recombinantprotein corresponding to the bHLH domain of da was supplied by Dr.Michael Caudy (Cornell University Medical College).

Mobility Shift Assay:

The oligonucleotide probes used for the electrophoretic mobility shiftassay (EMSA) are as follows: the Class A site used was the CACCTGhexamer (CAGGTG for the opposite strand) from the T5 promoter region ofthe Drosophila AS-C (Villares, R. et al. (1987) Cell 550:415-424)(5'-GATCGTAGTCACGCAGGTGGGATCCCTA-3' (SEQ ID NO:17) and5'-GATCTAGGGATCCCACCTGCGTGACTAC-3' (SEQ ID NO:18) for the oppositestrand), the Class B site was the CACGTG hexamer from the USF bindingsite in the adenovirus major late promoter (Gregor, P. D. et al. (1990)Genes & Dev. 4:1730-1740) (5'-GATCGGTGTAGGCCACGTGACCGGGTGT-3' (SEQ IDNO:19) and 5'-GATCACACCCGGTCACGTGGCCTACACC-3') (SEQ ID NO:20), the ClassC site was the CACGCG hexamer (CGCGTG for the opposite strand) from theAS-C T5 promoter (5'-GATCGGCAGCCGGCACGCGACAGGGCC-3' (SEQ ID NO:21) and5'-GATCGGCCCTGTCGCGTGCCGGCTGCC-3') (SEQ ID NO:22), and the N-box(CACNAG) was the double hexamer sequence from the Enhancer of splitE(spl)! m8 promoter (Klimbt, C. et al. (1989) EMBO J. 8:203-210)(5'-GATCACGCCACGAGCCACAAGGATTG-3' (SEQ ID NO:23) and5'-GATCCAATCCTTGTGGCTCGTGGCGT-3' (SEQ ID NO:24). One strand of theoligonucleotide was labeled with g-32P!ATP by T4 polynucleotide kinase,hybridized with three times excess of the opposite strand, and purifiedusing MERmaid oligonucleotide purification kit (BIO 101). 150 ng of theprotein was allowed to bind to 50,000 cpm (equivalent to 0.5 ng in atypical experiment) of the labeled probe for 15 min at room temperaturein 20 mM Hepes, pH 7.6, 50 mM KCl, 10 mM DTT, 5% glycerol, 0.5 mM EDTAand 0.3 mg/ml BSA. Two microgram of poly(dI-dC) was added to each 20 flreaction as on-specific DNA. Samples were analyzed on a 5% nativepolyacrylamide gel and visualized by autoradiography.

Regulation of CR8:

CR genes were originally defined in IL-2 stimulated normal human Tcells. To examine CR8 expression in cytokine-dependent cell lines, thelevel of CR8 expression was measured by Northern hybridization in theIL-2-dependent human T cell line Kit 225, the IL-3-dependent mouse pro-Bcell line Ba/F3, and the IL-2-dependent mouse T cell line CTLL2. Theresults of this experiment are illustrated in FIGS. 13A-13C. In FIG.13A, RNA was isolated from quiescent normal human T cells (lanes 1 and2), IL-2-dependent human T cell line Kit 225 (lanes 3 and 4),IL-3-dependent mouse pro-B cell line Ba/F3 (lanes 5 and 6) andIL-2-dependent mouse T cell line CTLL2 (lanes 7 and 8) left untreated(lanes 1, 3, 5, and 7) or stimulated with 500 pM recombinant human IL-2(lanes 2, 4, and 8) or 10 U/ml recombinant mouse IL-3 (lane 6) for 2 hrat 37° C. The amount of the growth factor used in the experiment wassufficient to induce maximal ³ H!thymidine incorporation. Ten microgramof total RNA was analyzed on formaldehyde/agarose gel and hybridizedwith either human (lanes 1 to 4) or mouse (lane 5 to 8) CR8 cDNA.

As shown in FIG. 13A, a single 3.2 kb species hybridized to the cDNAprobe, and in all three cell lines tested, the level of CR8 was clearlyaugmented when the cells were stimulated with their respective growthfactors. Correlation between the level of CR8 and that of DNA synthesiswas in the presence of growth-inhibitory agents was also examined. Inthis regard, increases in cytoplasmic cAMP are known to inhibit thegrowth of many cell types, including lymphocytes (Johnson, K. W. et al.(1988) Proc. Natl. Acad. Sci. USA 85:6072-6076). IFNs also exertantiproliferative activity on many cell types (Pestka, S. et al. (1987)Annu. Rev. Biochem. 56: 727-777). Therefore, Kit 225 was stimulated withIL-2, IFN β, or forskolin, which increases cytoplasmic cAMP byactivating adenylate cyclase, either alone or in combination.IL-2-dependent ³ H!thymidine incorporation was inhibited by IFNb andforskolin in Kit 225 cells in a dose-dependent fashion (FIG. 13B). FIG.13B demonstrates that IFNβ and forskolin inhibit IL-2-dependent ³H!thymidine incorporation by Kit 225 cells. Ten thousand quiescent Kit225 cells were incubated with indicated reagents in 200 fl for 24 hr at37° C. The culture was pulsed with ³ H!thymidine for the last 4 hr tomonitor the DNA synthesis. (.sup.•), IL-2 only (500 pM); (⋄), IL-2 500pM+varying concentrations of IFNb (U/ml); (o), IL-2 500 pM+varyingconcentrations of forskolin (fM). While forskolin was capable ofreducing the IL-2-dependent ³ H!thymidine incorporation almost to thebasal level, IFNβ-mediated inhibition never exceeded 70% of the maximalincorporation in several independent experiments. The expression of CR8was compared with that of c-myc, an IL-2-inducible immediate-early genethat encodes a bHLH protein and is implicated for cell proliferation(Marcu, K. B. et al. (1992) Annu. Rev. Biochem. 61:809-860).

FIG. 13C shows the effect of antiproliferative agents on the expressionof CR8 and c-myc transcripts in Kit 225. Quiescent Kit 225 cells wereleft untreated (lane 1) or incubated with 500 pM IL-2 (lane 2), 1000U/ml IFNb (lane 3), 100 fM forskolin (lane 4), 500 pM IL-2+1000 U/mlIFNa (lane 5) or 500 pM IL-2+100 fM forskolin (lane 6) for 2 hr at 37°C. and 15 fg of total RNA was analyzed. The same membrane was probedwith CR8 and c-myc. As shown in FIG. 13C, CR8 transcripts weremoderately induced, not only by IL-2, but also by IFNβ or forskolinalone. Furthermore, the simultaneous stimulation of quiescent Kit 225cells with IL-2 and IFNβ, or IL-2 and forskolin, did not suppress theIL-2-induced expression of CR8 transcripts. In contrast, IL-2-inductionof c-myc expression was substantially inhibited in the presence offorskolin, while IFNβ did not significantly reduce IL-2-promoted c-mycexpression.

Cloning of CR8:

The original human CR8 clone isolated from the thiol-selected libraryhad a 1.5 kb insert, while the full-length mRNA transcript was estimatedto be 3.2 kb from Northern blotting experiments. As the CR8 clone didnot have a long open reading frame, two full-length cDNA clones of humanCR8 were isolated from a λgt10 human T cell cDNA library after tworounds of screening with cDNA fragments of the CR8 clone. These twoclones were fully sequenced on both strands, and the amino acid sequencewas deduced (FIG. 14). In FIG. 14, the asterisk denotes the stop codon.In-frame termination codon in the 5'-untranslated region, the nucleotidesequences used for PCR and the polyadenylation signal are underlined.The amino acid residues in the bHLH region are double-underlined. Whenthe final consensus cDNA sequence of 2970 bp (excluding the poly(A)stretch) was screened against the nonredundant nucleotide databasesusing the NCBI BLAST E-mail server (GenBank release 86.0), no knowngenes in the database shared significant homology with CR8 except fornine EST sequences (Adams, M. D. et al. (1991) Science 252:1651-1656.).CR8 has an open reading frame of 412 amino acids, with an in-frametermination codon at position 198 followed by Met at position 240 in areasonable context for translation initiation (CGCCATGG) (Kozak, M.(1986) Cell 44:283-292). The MOTIFS program in the GCG package predictedthe presence of an HLH motif in CR8.

A mouse CR8 cDNA fragment corresponding to nt 388 to 2720 of the humansequence was also isolated from a λgt10 mouse cDNA library by comparisonof CR8 with other bHLH Proteins. The protein database search with theputative peptide sequence revealed that CR8 shares homology with thebHLH proteins encoded by Drosophila hairy gene and the enhancer of splitcomplex E(spl)-C! of neurogenic genes. FIGS. 15A-15B show a sequencecomparison of CR8 and other HLH proteins. Protein alignments were madeto maximize homology within the bHLH domain. Amino acids conserved amongmost HLH proteins are shaded. The proline residues in the basic regionand the arginine residues at position 13 ("R13") are boxed. The boxedalanine residue in MyoD is the one whose substitution to prolineabrogated the DNA binding and muscle-specific gene activation activityof MyoD (Davis, R. L. et al. (1990) Cell 60:733-746). h!, human; D!,Drosophila melanogaster; r!, rat; and m!, mouse. Sources for sequences:hairy, (Rushlow, C. A. et al. (1989) EMBO J. 8:3095-3103); Enhancer ofsplit E(spl)!m7, (Klimbt, C. et al. (1989) EMBO J. 8:203-210); deadpan(dpn), (Bier, E. et al. (1992) Genes & Dev 6:2137-2151); HES-1, (Sasai,Y. et al. (1992) Genes & Dev 6:2620-2634); daughterless (da), (Caudy, M.et al. (1988) Cell 55:1061-1067); E12 and E47, (Murre, C. et al. (1989)Cell 56:777-783); MyoD, (Davis, R. L. et al. (1987) Cell 51:987-1000);Tal-1, (Begley, C. G. et al. (1989) Proc. Natl. Acad. Sci. USA86:10128-10132); USF, (Gregor, P. D. et al. (1990) Genes & Dev4:1730-1740); Max, (Blackwood, E. M. et al. (1991) Science251:1211-1217); N-myc, (Slamon, D. J. et al. (1986) Science232:768-772); L-myc, (Kaye, F. et al. (1988) Mol. Cell. Biol.8:186-195); c-myc, (Gazin, C. et al. (1984) EMBO J. 3:383-387);extramacrochaetae (emc), (Ellis, H. M. et al. (1990) Cell 61:27-38;Garrell, J. et al. (1990) Cell 61:39-48) and Id1, (Benezra, R. et al.(1990) Cell 61:49-59).

When CR8 was aligned with other bHLH proteins (FIGS. 15A-15B), it wasclear that most of the residues conserved throughout the family werepresent in CR8. Taken together with the result of the MOTIFS program, itwas concluded that CR8 is a bHLH protein. The amino acid sequence of the58-residue bHLH domain of CR8 showed 40% identity to hairy, 41% toE(spl)m7, and 45% to one of their mammalian counterparts HES-1. Thisdegree of amino acid identity accounts well for the failure to detectany significant homology to any known bHLH proteins at the nucleotidesequence level. The amino acid sequence for human and mouse CR8 was 100%identical in the bHLH domain.

FIG. 15B shows a sequence comparison of CR8 and hairy-related bHLH.Conserved amino acids are shaded. Note that HES-2, 3 and 5 proteins donot align perfectly in the hairy-related homology region (HRHR)-2.Sources for sequences: HES-2, (Ishibashi, M. et al. (1993) Eur. J.Biochem. 215:645-652); HES-3, (Sasai, Y. et al. (1992) Genes & Dev6:2620-2634); HES-5, (Akazawa, C. et al. (1992) J. Biol. Chem.267:21879-21885); human hairy-like (HHL), (Feder, J. N. et al. (1994)Genomics 20:56-61); Drosophila melanogaster hairy h(m)!, (Rushlow, C. A.et al. (1989) EMBO J. 8:3095-3103); Drosophila virilis hairy h(v)!,(Wainwright, S. M. et al. (1992) Mol. Cell. Biol. 12:2475-2483);Tribolium hairy h(T)!, (Sommer, R. J. et al. (1993) Nature 361:448-450);E(spl)m5 and m8, (Klimbt, C. et al. (1989) EMBO J. 8:203-210); E(spl)m3,b/A, g/B, and d/C, (Deldakis, C. et al. (1992) Proc. Natl. Acad. Sci.USA 89:8731-8735; Knust, E. et al. (1992) Genetics 132:505-518). Asshown in FIG. 15B, the amino acid sequence of the bHLH region of CR8 isaligned with hairy, bHLH proteins of the E(spl)-C, deadpan (dpn) andtheir mammalian homologs (the term "hairy-related bHLH proteins" referto them collectively). Among all the bHLH proteins described thus far,CR8 is the only one with a proline residue in the basic region, otherthan the hairy-related bHLH proteins. However, while the position of theproline residue is strictly conserved throughout the hairy-related bHLHproteins, in CR8 it is offset N-terminally by two residues. CR8 andhairy-related bHLH proteins are different in the C-terminus as well; allthe hairy-related bHLH proteins terminate with a specificTrp-Arg-Pro-Trp (WRPW) motif, which is absent in CR8. Nevertheless, CR8showed appreciable homology to other hairy-related bHLH proteins in theregion immediately C-terminal to the bHLH domain, which has been shownpreviously to be rich in hydrophobic residues, and proposed to form twomore α-helices in bHLH proteins of the E(spl)-C (43). This region isreferred to herein as the "hairy-related homology region (HRHR)-2", theHRHR-1 being the bHLH domain. The region N-terminal to the bHLH domainand the C-terminal half of the CR8 protein are rich in proline (8proline residues between positions 1 and 30, 22 between 310 and 405).Notably, there are no known proteins in the data bases that sharehomologies to these most N-terminal and C-terminal regions of CR8.

Tissue Distribution of CR8 Transcripts:

Murre et al. ((1989) Cell 58:537-544) categorized bHLH proteins basedupon their tissue distribution. While proteins such as MyoD and AS-Cgene products show a cell-type specific expression, others such asE12/E47 and da are fairly ubiquitously expressed. The tissuedistribution of CR8 was analyzed using a Multiple Tissue Northern blot.CR8 transcripts of the expected size (3.2 kb) were detected in alltissues examined except placenta (see FIG. 16). FIG. 16 demonstratesthat the Multiple Tissue Northern Blot membranes (Clontech; each lanecontains 2 fg poly(A)+ RNA from indicated human tissue) were hybridizedwith human CR8 probe.

The expression of CR8 in peripheral blood leukocytes was unexpected, inthat CR8 is not expressed by quiescent T cells. This may reflect muchhigher sensitivity of Multiple Tissue Northern blot prepared frompoly(A)+ RNA compared to our previous Northern blots, which used totalRNA. Alternatively, the contribution of other leukocytes such as Bcells, NK cells, monocytes and granulocytes that were not present in theoriginal T cell preparations could account for CR8 expression by theperipheral blood leukocytes.

DNA-Binding Activity of CR8:

The canonical bHLH binding sequence is called the E-box, CANNTG,originally identified in the immunoglobulin heavy chain enhancer(Ephrussi, A. et al. (1985) Science 227:134-140). Many bHLH proteinswere later divided into two mutually exclusive classes, depending onwhether they bind to the Class A sites (CAGCTG/CACCTG) or the Class Bsites (CACGTG/CATGTG) (Dang, C. V. et al. (1992) Proc. Natl. Acad. Sci.USA 89:599-602). The presence of an arginine residue at position 13("R13", see FIG. 15A) in the basic region, which CR8 contains, isconsidered to be the key structural criterion that defines Class Bbinding specificity. However, despite the presence of "R13",hairy-related bHLH proteins are reported to prefer noncanonical bindingsites such as the N-box (CACNAG) (Akazawa, C. et al. (1992) J. Biol.Chem. 267:21879-21885; Sasai, Y. et al. (1992) Genes & Dev 6:2620-2634;Tietze, K. et al. (1992) Proc. Natl. Acad. Sci. USA 89:6152-6156) or theClass C (CACGCG) sites (Ohsako, S. et al. (1994) Genes & Dev8:2743-2755; Van Doren, M. et al. (1994) Genes & Dev 8:2729-2742.).Therefore, the binding of CR8 to all of these sites was tested.

Since it is well documented that the bHLH domain is sufficient todetermine its DNA binding specificity (Pognonec, P. et al. (1994) Mol.Cell. Biol. 11:5125-5136), the bHLH domain of CR8 (CR8 bHLH) expressedin E. coli was employed for this study. A histidine-tag was added tofacilitate the purification of the recombinant protein. While most ofthe recombinant protein localized in inclusion bodies, there was stillenough soluble protein in the cytoplasm, thereby enabling itspurification under native conditions using a Ni²⁺ column. A single bandof protein was detected at the expected size (16.6 kD with thehistidine-tag) by silver staining. EMSA was carried out using thisrecombinant protein.

FIG. 17A is an EMSA shows binding of recombinant bHLH proteins to theradiolabeled probes. CR8 bHLH protein strongly binds to the Class B(CACGTG, lane 3) and the Class C (CACGCG, lane 4) sites, and weakly tothe N box (CACNAG, lane 5) sequence but not to the Class A (CACCTG, lane2) site. Binding of the bHLH region of da protein to the Class A site isshown as control (lane 1). As shown in FIG. 17A, CR8 bHLH bound to theClass B and the Class C probes, but only weakly to the N-box probe, andnot at all to the Class A probe. However, the control da bHLH proteineffectively recognized and bound to the same Class A probe.

To examine the relative binding affinity, a large excess of non-labeledoligonucleotide was added to the reaction as competitor. FIG. 17B showscompetition of the binding of CR8 bHLH to the Class B sites. 0.5 ng ofthe radiolabeled Class B probe was incubated with CR8 bHLH in theabsence (lane 1) or the presence (lanes 2 to 7) of either 25 ng (50-foldexcess; lanes 2, 4 and 6) and 250 ng (500-fold excess; lanes 3, 5 and 7)of unlabeled competitors. FIG. 17B demonstrates that the binding of CR8bHLH to the radiolabeled Class B site can be abolished partially by a50-fold excess, and completely by a 500-fold excess of Class B site(lanes 2 and 3), while a 500-fold excess of Class C site only partiallydisplaced CR8 bHLH from the labeled Class B probe (lanes 4 and 5) andthe N-box sequence did not affect the binding at all (lanes 6 and 7).Thus, since all these experiments were done in the absence of other HLHproteins, it appears that CR8 bHLH bound to the Class B sequence as ahomodimer with the highest affinity.

The CR8 gene encodes a novel bHLH protein that appears to fit into aclass by itself. Other than c-myc, CR8 is the first bHLH-containingprotein found to be induced by cytokines. Also, from its predicted aminoacid sequence, CR8 clearly contains a bHLH motif most closely related tothe hairy family, but the amino acid sequence of the basic regiondiffers from other hairy-related proteins: the position of the prolineresidue is N-terminal to the defining proline of the hairy-relatedproteins, and CR8 lacks the C-terminal WRPW sequence found in all otherhairy-related-related proteins. These differences in the amino acidsequence, especially of the basic region, most likely account for theunique binding specificity of the CR8 bHLH domain. Instead of preferringClass C sites according to the other hairy-related family members(Ohsako, S. et al. (1994) Genes & Dev 8:2743-275572; Van Doren, M. etal. (1994) Genes & Dev 8:2729-2742), CR8 binds preferentially to Class Bsites.

The identification of CR8 as a bHLH protein, thereby functioning, mostlikely, as a regulator of subsequent gene expression stimulated by IL-2,provides a link between the immediate biochemical events triggered bycytokine receptors and the subsequent events of proliferation and/ordifferentiation. Thus far, IL-2 has been found to activate theserine/threonine kinase proto-oncogene Raf-1 (Turner, B. et al. (1991)Proc. Natl. Acad. Sci. USA 88:1227-1231;Zmuidzinas, A. et al. (1991)Mol. Cell Biol. 11:2794-2803) and the tyrosine-specific kinases JAK 1and JAK 3 (Beadling, C. et al. (1994) EMBO J. 13:5605-5615; Miyazaki, T.et al. (1994) Science 266:1045-1047; Russell, S. M. et al. (1994)Science 266:1042-1045).

From the results described herein comparing the effects of IFNa andforskolin on CR8 and c-myc gene expression, the regulation of these twobHLH genes is clearly distinct. It is also of interest that althoughIFNβ antagonizes IL-2-promoted cell cycle progression, it promotes theexpression of both CR8 and c-myc. Indeed, induction of c-myc by IFNβ wasunexpected, as it was previously reported to be suppressed by IFNs(Einat, M. et al. (1985) Nature 313:597-600). The bHLH region of CR8 ismost homologous to that of hairy and the bHLH proteins of the E(spl)-C.In Drosophila, the hairy-related bHLH proteins function astranscriptional repressors, and this activity requires the basic DNAbinding region, as well as the interaction with a non-HLH protein termedgroucho (gro) via the C-terminal WRPW motif (Paroush, Z. et al. (1994)Cell 79:805-815). Although mammalian homologues of gro have beenidentified (Stifani, S. et al. (1992) Nat. Genet. 2:119-127), they arenot likely to interact with CR8 because CR8 lacks the WRPW motif.

The results described herein indicate that CR8 recombinant protein bindsto Class B E-box sites as a homodimer. This result is consistent withthe predictions from DNA-bHLH protein co-crystals (Ferr-D'Amar, A. R. etal. (1994) EMBO J. 13:180-189; Ferr-D'Amar, A. R. et al. (1993) Nature363:38-45). However, it is noteworthy in that CR8 is the first bHLHvertebrate protein without a leucine zipper (LZ) motif found to bindClass B sites. Protein dimerization is more selective than DNA binding,but currently no rules are available that predict the dimerizationpreference of any given HLH proteins. Even so, a Class A-binding proteinseems to form DNA binding heterodimers only with other Class A proteins,and a bHLH protein with a LZ does not form heterodimers with thosewithout LZs (Blackwood, E. M. et al. (1991) Science 251:1211-1217;Prendergast, G. C. et al. (1991) Cell 65:395-407). Therefore, if CR8does form heterodimers, the most likely partner is a class B-bindingbHLH protein without a LZ.

Although CR8 is most homologous to hairy in its bHLH domain, itspreference for Class B E-box binding sites rather than class C sites,and its lack of a C-terminal WPRW motif, clearly sets CR8 apart and doesnot predict necessarily that CR8 may act as a transcriptional repressoras do hairy-related proteins. Recently, Id proteins that lack a basicregion have been shown to favor proliferation, presumably by formingheterodimers with differentiation inducing bHLH proteins, therebypreventing DNA binding and transcriptional activation of genes thatprogram differentiation (Barone, M. V. et al. (1994) Proc. Natl. Acad.Sci. USA 91:4985-4988; Hara, E. et al. (1994) J. Biol. Chem.269:2139-2145; Iavarone, A. et al. (1994) Genes & Dev 1270-1284).Therefore, CR8 could promote proliferation by suppressingdifferentiation by either of these transcriptional repressor mechanisms.Alternatively, CR8 could also activate transcription like the bHLH-LZMyc family.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES:35    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 2406 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 116..722    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    AACCCAACCGCAGTTGACTAGCACCTGCTACCGCGCCTTTGCTTCCTGGCGCACGCGGAG60    CCTCCTGGAGCCTGCCACCATCCTGCCTACTACGTGCTGCCCTGCGCCCGCAGCCATG118    TGCCGCACCCTGGCCGCCTTCCCCACCACCTGCCTGGAGAGAGCCAAA166    GAGTTCAAGACACGTCTGGGGATCTTTCTTCACAAATCAGAGCTGGGC214    TGCGATACTGGGAGTACTGGCAAGTTCGAGTGGGGCAGTAAACACAGC262    AAAGAGAATAGAAACTTCTCAGAAGATGTGCTGGGGTGGAGAGAGTCG310    TTCGACCTGCTGCTGAGCAGTAAAAATGGAGTGGCTGCCTTCCACGCT358    TTCCTGAAGACAGAGTTCAGTGAGGAGAACCTGGAGTTCTGGCTGGCC406    TGTGAGGAGTTCAAGAAGATCCGATCAGCTACCAAGCTGGCCTCCAGG454    GCACACCAGATCTTTGAGGAGTTCATTTGCAGTGAGGCCCCTAAAGAG502    GTCAACATTGACCATGAGACCCGCGAGCTGACGAGGATGAACCTGCAG550    ACTGCCACAGCCACATGCTTTGATGCGGCTCAGGGGAAGACACGTACC598    CTGATGGAGAAGGACTCCTACCCACGCTTCCTGAAGTCGCCTGCTTAC646    CGGGACCTGGCTGCCCAAGCCTCAGCCGCCTCTGCCACTCTGTCCAGC694    TGCAGCCTGGACCAGCCCTCACACACCTGAGTCTCCACGGCAGTGAGG742    AAGCCAGCCGGGAAGAGAGGTTGAGTCACCCATCCCCGAGGTGGCTGCCCCTGTGTGGGA802    GGCAGGTTCTGCAAAGCAAGTGCAAGAGGACAAAAAAAAAAAAAAAAAAAAAAAATGCGC862    TCCAGCAGCCTGTTTGGGAAGCAGCAGTCTCTCCTTCAGATACTGTGGGACTCATGCTGG922    AGAGGAGCCGCCCACTTCCAGGACCTGTGAATAAGGGCTAATGATGAGGGTTGGTGGGGC982    TCTCTGTGGGGCAAAAAGGTGGTATGGGGGTTAGCACTGGCTCTCGTTCTCACCGGAGAA1042    GGAAGTGTTCTAGTGTGGTTTAGGAAACATGTGGATAAAGGGAACCATGAAAATGAGAGG1102    AGGAAAGACATCCAGATCAGCTGTTTTGCCTGTTGCTCAGTTGACTCTGATTGCATCCTG1162    TTTTCCTAATTCCCAGACTGTTCTGGGCACGGAAGGGACCCTGGATGTGGAGTCTTCCCC1222    TTTGGCCCTCCTCACTGGCCTCTGGGCTAGCCCAGAGTCCCTTAGCTTGTACCTCGTAAC1282    ACTCCTGTGTGTCTGTCCAGCCTTGCAGTCATGTCAAGGCCAGCAAGCTGATGTGACTCT1342    TGCCCCATGCGAGATATTTATACCTCAAACACTGGCCTGTGAGCCCTTTCCAAGTCAGTG1402    GAGAGCCCTGAAAGGAGCCTCACTTGAATCCAGCTCAGTGCTCTGGGTGGCCCCCTGCAG1462    GTGCCCCCTGACCCTGCGTTGCAGCAGGGTCCACCTGTGAGCAGGCCCGCCCTGGGCCCT1522    CTTCCTGGATGTGCCCTCTCTGAGTTCTGTGCTGTCTCTTGGAGGCAGGGCCCAGGAGAA1582    CAAAGTGTGGAGGCCTCGGGGAGTGACTTTTCCAGCTCTCATGCCCCGCAGTGTGGAACA1642    AGGCAGAAAAGGATCCTAGGAAATAAGTCTCTTGGCGGTCCCTGAGAGTCCTGCTGAAAT1702    CCAGCCAGTGTTTTTTGTGGTATGAGAACAGCCAAAAAGAGATGCCCCGAGATAGAAGGG1762    GAGCCTTGTGTTTCTTTCCTGCAGACGTGAGATGAACACTGGAGTGGGCAGAGGTGGCCC1822    AGGACCATGACACCCTTAGAGTGCAGAAGCTGGGGGGAGAGGCTGCTTCGAAGGGCAGGA1882    CTGGGGATAATCAGAACCTGCCTGTCACCTCAGGGCATCACTGAACAAACATTTCCTGAT1942    GGGAACTCCTGCGGCAGAGCCCAGGCTGGGGAAGTGAACTACCCAGGGCAGCCCCTTTGT2002    GGCCCAGGATAATCAACACTGTTCTCTCTGTACCATGAGCTCCTCCAGGAGATTATTTAA2062    GTGTATTGTATCATTGGTTTTCTGTGATTGTCATAACATTGTTTTTGTTACTGTTGGTGC2122    TGTTGTTATTTATTATTGTAATTTCAGTTTGCCTCTACTGGAGAATCTCAGCAGGGGTTT2182    CAGCCTGACTGTCTCCCTTTCTCTACCAGACTCTACCTCTGAATGTGCTGGGAACCTCTT2242    GGAGCCTGTCAGGAACTCCTCACTGTTTAAATATTTAGGTATTGTGACAAATGGAGCTGG2302    TTTCCTAGAAATGAATGATGTTTGCAATCCCCATTTTCCTGTTTCAGCATGTTATATTCT2362    TATGAAATAAAAGCCCAAGTCCAATATGAAAAAAAAAAAAAAAA2406    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 202 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    MetCysArgThrLeuAlaAlaPheProThrThrCysLeuGluArgAla    151015    LysGluPheLysThrArgLeuGlyIlePheLeuHisLysSerGluLeu    202530    GlyCysAspThrGlySerThrGlyLysPheGluTrpGlySerLysHis    354045    SerLysGluAsnArgAsnPheSerGluAspValLeuGlyTrpArgGlu    505560    SerPheAspLeuLeuLeuSerSerLysAsnGlyValAlaAlaPheHis    65707580    AlaPheLeuLysThrGluPheSerGluGluAsnLeuGluPheTrpLeu    859095    AlaCysGluGluPheLysLysIleArgSerAlaThrLysLeuAlaSer    100105110    ArgAlaHisGlnIlePheGluGluPheIleCysSerGluAlaProLys    115120125    GluValAsnIleAspHisGluThrArgGluLeuThrArgMetAsnLeu    130135140    GlnThrAlaThrAlaThrCysPheAspAlaAlaGlnGlyLysThrArg    145150155160    ThrLeuMetGluLysAspSerTyrProArgPheLeuLysSerProAla    165170175    TyrArgAspLeuAlaAlaGlnAlaSerAlaAlaSerAlaThrLeuSer    180185190    SerCysSerLeuAspGlnProSerHisThr    195200202    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1223 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 171..351    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    ATTTAGAGCAACTCAGGAAATAGGTGCACACAAGCAAACCATGTGGTTAAAGCCTTTGGA60    ACTGGTTTGAGCAAAGCTGTAGGTGATTTGACAAAATCATCTGCAAAACCAGATTTCTAA120    CACCTCCCTGCTGTGTATCTCATTTCTGCTGATGTGTGGTGCTTCATAAGATGGGG176    ACGTTAAGCATGCAGCAACTACAGTCATTTGTTCTCAGAGGTCTGGAC224    CAAAGAGAAACAAGAAAAGCTGGAGTCACACTACCAAAGGCCGAAGCT272    GAGCAACAGAGCTCTGGAGTCAGCTGCCTGGGTTCAGCATGCAGCGCT320    GCCGTGGACGATCTGTCTCTCTTGCATATATGACTTACCAGTTTTACTTTC371    AGTCTCTCCATTTCTAATTAAATGAGATGCAGAAATGCTGGTGCCTTGCTATGATGTTTG431    CAGTTATTATTTCTAGGAAAAAAAATATTATTGTTACTCAGTATCTGGTCTAGCTACTTG491    GACAACTGGACTATCCCCCTCCTTTCAAGGGAGGGCAAAGCATTTCAGAAAAGAACTAAG551    TGCTATTTCTCTGCTTCAGGAATGTCTCCCGTATGTAAAAGAATGTGGCTTCAGGGAGTA611    GCATGTGTTGTAAAGGTGGATGGGTCTAACTTCATGGACAGCTCTGACATCCACTAGCTA671    TGCCACCTGATGCAAACCACTTGGGCTGTCTGCAGTTTCGTTTATCTTTCTGGAATTGGT731    AATAACAACCACCTGGCAAGATCACTGTTATGAATACGGAGGATCAAAGTTGTGAAGTTA791    TTTTGTAAAGTGAAATGTTCTGAAAAATGGATTTTAACAGTGTCAGCGAAAAGTAGATTT851    TTGACATTTATCAAGAGTTCAGCTAATGAAAACAAGTATGGATAATAGTTACATAGAACT911    GTCTACTTTACTCAGTACTTTAGCATATGCTATTATATTTAATCTTCTTAAAAAGTAGGA971    AATTATACAAGCCATGTATTGATATTATTGTGGTGGTTGTCGTTCTCAATTACACACTGA1031    ATATTAAGACCTCTCAGGTAGCAGCTGGAAGGACATTGTATCCAGTTTCCTGATTGTTTT1091    CAATGGAATAATCATGTATACATGCACTACTAATGAGACAATGGTGATTCTAAAAGCTTA1151    ATCAGGGGGACTTTTGTGTATTCCAAATCTACTAAAAATAAAGAAACACAGAAATGAGAA1211    AAAAAAAAAAAA1223    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 60 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    MetGlyThrLeuSerMetGlnGlnLeuGlnSerPheValLeuArgGly    151015    LeuAspGlnArgGluThrArgLysAlaGlyValThrLeuProLysAla    202530    GluAlaGluGlnGlnSerSerGlyValSerCysLeuGlySerAlaCys    354045    SerAlaAlaValAspAspLeuSerLeuLeuHisIle    505560    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 2450 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 229..1303    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    CGCGGGAGCCTCGAGCGCCGCTCGGATGCAGAAGCCGAGCCGCCACTCGGCGCGCGGTGG60    GAGACCCAGGGCAAGCCGCCGTCGGCGCGCTGGGTGCGGGAAGGGGGCTCTGGATTTCGG120    TCCCTCCCCTTTTTCCTCTGAGTCTCGGAACGCTCCAGATCTCAGACCCTCTTCCTCCCA180    GGTAAAGGCCGGGAGAGGAGGGCGCATCTCTTTTCCAGGCACCCCACCATGGGAAAT237    GCCTCCAATGACTCCCAGTCTGAGGACTGCGAGACGCGACAGTGGTTT285    CCCCCAGGCGAAAGCCCAGCCATCAGTTCCGTCATGTTCTCGGCCGGG333    GTGCTGGGGAACCTCATAGAACTGGCGCTGCTGGCGCGCCGCTGGCAG381    GGGGACGTGGGGTGCAGCGCCGGCCGTAGGAGCTCCCTCTCCTTGTTC429    CACGTGCTGGTGACCGAGCTGGTGTTCACCGACCTGCTCGGGACCTGC477    CTCATCAGCCCAGTGGTACTGGCTTCGTACGCGCGGAACCAGACCCTG525    GTGGCACTGGCGCCCGAGAGCCGCGCGTCCACCTACTTCGCTTTCGCC573    ATGACCTTCTTCAGCCTGGCCACGATGCTCATGCTCTTCACCATGGCC621    CTGGAGCGCTACCTCTCGATCGGGCACCCCTACTTCTACCAGCGCCGC669    GTCTCGCGCTCCGGGGGCCTGGCCGTGCTGCCTGTCATCTATGCAGTC717    TCCCTGCTCTTCTGCTCACTGCCGCTGCTGGACTATGGGCAGTACGTC765    CAGTACTGCCCCGGGACCTGGTGCTTCATCCGGCACGGGCGGACCGCT813    TACCTGCAGCTGTACGCCACCCTGCTGCTGCTTCTCATTGTCTCGGTG861    CTCGCCTGCAACTTCAGTGTCATTCTCAACCTCATCCGCATGCACCGC909    CGAAGCCGGAGAAGCCGCTGCGGACCTTCCCTGGGCAGTGGCCGGGGC957    GGCCCCGGGGCCCGCAGGAGAGGGGAAAGGGTGTCCATGGCGGAGGAG1005    ACGGACCACCTCATTCTCCTGGCTATCATGACCATCACCTTCGCCGTC1053    TGCTCCTTGCCTTTCACGATTTTTGCATATATGAATGAAACCTCTTCC1101    CGAAAGGAAAAATGGGACCTCCAAGCTCTTAGGTTTTTATCAATTAAT1149    TCAATAATTGACCCTTGGGTCTTTGCCATCCTTAGGCCTCCTGTTCTG1197    AGACTAATGCGTTCAGTCCTCTGTTGTCGGATTTCATTAAGAACACAA1245    GATGCAACACAAACTTCCTGTTCTACACAGTCAGATGCCAGTAAACAG1293    GCTGACCTTTGAGGTCAGTAGTTTAAAAGTTCTTAGTTATATAGCATCTG1343    GAAGATCATTTTGAAATTGTTCCTTGGAGAAATGAAAACAGTGTGTAAACAAAATGAAGC1403    TGCCCTAATAAAAAGGAGTATACAAACATTTAAGCTGTGGTCAAGGCTACAGATGTGCTG1463    ACAAGGCACTTCATGTAAAGTGTCAGAAGGAGCTACAAAACCTACCCTCAGTGAGCATGG1523    TACTTGGCCTTTGGAGGAACAATCGGCTGCATTGAAGATCCAGCTGCCTATTGATTTAAG1583    CTTTCCTGTTGAATGACAAAGTATGTGGTTTTGTAATTTGTTTGAAACCCCAAACAGTGA1643    CTGTACTTTCTATTTTAATCTTGCTACTACCGTTATACACATATAGTGTACAGCCAGACC1703    AGATTAAACTTCATATGTAATCTCTAGGAAGTCAATATGTGGAAGCAACCAAGCCTGCTG1763    TCTTGTGATCACTTAGCGAACCCTTTATTTGAACAATGAAGTTGAAAATCATAGGCACCT1823    TTTACTGTGATGTTTGTGTATGTGGGAGTACTCTCATCACTACAGTATTACTCTTACAAG1883    AGTGGACTCAGTGGGTTAACATCAGTTTTGTTTACTCATCCTCCAGGAACTGCAGGTCAA1943    GTTGTCAGGTTATTTATTTTATAATGTCCATATGCTAATAGTGATCAAGAAGACTTTAGG2003    AATGGTTCTCTCAACAAGAAATAATAGAAATGTCTCAAGGCAGTTAATTCTCATTAATAC2063    TCTTTATCCTATTTCTGGGGGAGGATGTACGTGGCCATGTATGAAGCCAAATATTAGGCT2123    TAAAAACTGAAAAATCTGGTTCATTCTTCAGATATACTGGAACCCTTTTAAAGTTGATAT2183    TGGGGCCATGAGTAAAATAGATTTTATAAGATGACTGTGTTGTACTAAAATTCATCTGTC2243    TATATTTTATTTAGGGGACATGGTTTGACTCATCTTATATGGGAAACCATGTAGCAGTGA2303    GTCATATCTTAATATATTTCTAAATGTTTGGCATGTAAACGTAAACTCAGCATCACAATA2363    TTTCAGTGAATTTGCACTGTTTAATCATAGTTACTGTGTAAACTCATCTGAAATGTTACC2423    AAAAATAAACTATAAAACAAAATTTGA2450    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 358 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    MetGlyAsnAlaSerAsnAspSerGlnSerGluAspCysGluThrArg    151015    GlnTrpPheProProGlyGluSerProAlaIleSerSerValMetPhe    202530    SerAlaGlyValLeuGlyAsnLeuIleGluLeuAlaLeuLeuAlaArg    354045    ArgTrpGlnGlyAspValGlyCysSerAlaGlyArgArgSerSerLeu    505560    SerLeuPheHisValLeuValThrGluLeuValPheThrAspLeuLeu    65707580    GlyThrCysLeuIleSerProValValLeuAlaSerTyrAlaArgAsn    859095    GlnThrLeuValAlaLeuAlaProGluSerArgAlaSerThrTyrPhe    100105110    AlaPheAlaMetThrPhePheSerLeuAlaThrMetLeuMetLeuPhe    115120125    ThrMetAlaLeuGluArgTyrLeuSerIleGlyHisProTyrPheTyr    130135140    GlnArgArgValSerArgSerGlyGlyLeuAlaValLeuProValIle    145150155160    TyrAlaValSerLeuLeuPheCysSerLeuProLeuLeuAspTyrGly    165170175    GlnTyrValGlnTyrCysProGlyThrTrpCysPheIleArgHisGly    180185190    ArgThrAlaTyrLeuGlnLeuTyrAlaThrLeuLeuLeuLeuLeuIle    195200205    ValSerValLeuAlaCysAsnPheSerValIleLeuAsnLeuIleArg    210215220    MetHisArgArgSerArgArgSerArgCysGlyProSerLeuGlySer    225230235240    GlyArgGlyGlyProGlyAlaArgArgArgGlyGluArgValSerMet    245250255    AlaGluGluThrAspHisLeuIleLeuLeuAlaIleMetThrIleThr    260265270    PheAlaValCysSerLeuProPheThrIlePheAlaTyrMetAsnGlu    275280285    ThrSerSerArgLysGluLysTrpAspLeuGlnAlaLeuArgPheLeu    290295300    SerIleAsnSerIleIleAspProTrpValPheAlaIleLeuArgPro    305310315320    ProValLeuArgLeuMetArgSerValLeuCysCysArgIleSerLeu    325330335    ArgThrGlnAspAlaThrGlnThrSerCysSerThrGlnSerAspAla    340345350    SerLysGlnAlaAspLeu    355358    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 2946 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 215..2503    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    GGGGGGAAAGGAAAATAATACAATTTCAGGGGAAGTCGCCTTCAGGTCTGCTGCTTTTTT60    ATTTTTTTTTTTTTAATTAAAAAAAAAAAGGACATAGAAAACATCAGTCTTGAACTTCTC120    TTCAAGAACCCGGGCTGCAAAGGAAATCTCCTTTGTTTTTGTTATTTATGTGCTGTCAAG180    TTTTGAAGTGGTGATCTTTAGACAGTGACTGAGTATGGATCATTTGAACGAG232    GCAACTCAGGGGAAAGAACATTCAGAAATGTCTAACAATGTGAGTGAT280    CCGAAGGGTCCACCAGCCAAGATTGCCCGCCTGGAGCAGAACGGGAGC328    CCGCTAGGAAGAGGAAGGCTTGGGAGTACAGGTGCAAAAATGCAGGGA376    GTGCCTTTAAAACACTCGGGCCATCTGATGAAAACCAACCTTAGGAAA424    GGAACCATGCTGCCAGTTTTCTGTGTGGTGGAACATTATGAAAACGCC472    ATTGAATATGATTGCAAGGAGGAGCATGCAGAATTTGTGCTGGTGAGA520    AAGGATATGCTTTTCAACCAGCTGATCGAAATGGCATTGCTGTCTCTA568    GGTTATTCACATAGCTCTGCTGCCCAGGCCAAAGGGCTAATCCAGGTT616    GGAAAGTGGAATCCAGTTCCACTGTCTTACGTGACAGATGCCCCTGAT664    GCTACAGTAGCAGATATGCTTCAAGATGTGTATCATGTGGTCACATTG712    AAAATTCAGTTACACAGTTGCCCCAAACTAGAAGACTTGCCTCCCGAA760    CAATGGTCGCACACCACAGTGAGGAATGCTCTGAAGGACTTACTGAAA808    GATATGAATCAGAGTTCATTGGCCAAGGAGTGCCCCCTTTCACAGAGT856    ATGATTTCTTCCATTGTGAACAGTACTTACTATGCAAATGTCTCAGCA904    GCAAAATGTCAAGAATTTGGAAGGTGGTACAAACATTTCAAGAAGACA952    AAAGATATGATGGTTGAAATGGATAGTCTTTCTGAGCTATCCCAGCAA1000    GGCGCCAATCATGTCAATTTTGGCCAGCAACCAGTTCCAGGGAACACA1048    GCCGAGCAGCCTCCATCCCCTGCGCAGCTCTCCCATGGCAGCCAGCCC1096    TCTGTCCGGACACCTCTTCCAAACCTGCACCCTGGGCTCGTATCAACA1144    CCTATCAGTCCTCAATTGGTCAACCAGCAGCTGGTGATGGCTCAGCTG1192    CTGAACCAGCAGTATGCAGTGAATAGACTTTTAGCCCAGCAGTCCTTA1240    AACCAACAATACTTGAACCACCCTCCCCCTGTCAGTAGATCTATGAAT1288    AAGCCTTTGGAGCAACAGGTTTCGACCAACACAGAGGTGTCTTCCGAA1336    ATCTACCAGTGGGTACGCGATGAACTGAAACGAGCAGGAATCTCCCAG1384    GCGGTATTTGCACGTGTGGCTTTTAACAGAACTCAGGGCTTGCTTTCA1432    GAAATCCTCCGAAAGGAAGAGGACCCCAAGACTGCATCCCAGTCTTTG1480    CTGGTAAACCTTCGGGCTATGCAGAATTTCTTGCAGTTACCGGAAGCT1528    GAAAGAGACCGAATATACCAGGACGAAAGGGAAAGGAGCTTGAATGCT1576    GCCTCGGCCATGGGTCCTGCCCCCCTCATCAGCACACCACCCAGCCGT1624    CCTCCCCAGGTGAAAACAGCTACTATTGCCACTGAAAGGAATGGGAAA1672    CCAGAGAACAATACCATGAACATTAATGCTTCCATTTATGATGAGATT1720    CAGCAGGAAATGAAGCGTGCTAAAGTGTCTCAAGCACTGTTTGCAAAG1768    GTTGCAGCAACCAAAAGCCAGGGATGGTTGTGCGAGCTGTTACGCTGG1816    AAAGAAGATCCTTCTCCAGAAAACAGAACCCTGTGGGAGAACCTCTCC1864    ATGATCCGAAGGTTCCTCAGTCTTCCTCAGCCAGAACGTGATGCCATT1912    TATGAACAGGAGAGCAACGCGGTGCATCACCATGGCGACAGGCCGCCC1960    CACATTATCCATGTTCCAGCAGAGCAGATTCAGCAACAGCAGCAGCAA2008    CAGCAACAGCAGCAGCAGCAGCAGCAGGCACCGCCGCCTCCACAGCCA2056    CAGCAGCAGCCACAGACAGGCCCTCGGCTCCCCCCACGGCAACCCACG2104    GTGGCCTCTCCAGCAGAGTCAGATGAGGAAAACCGACAGAAGACCCGG2152    CCACGAACAAAAATTTCAGTGGAAGCCTTGGGAATCCTCCAGAGTTTC2200    ATACAAGACGTGGGCCTGTACCCTGACGAAGAGGCCATCCAGACTCTG2248    TCTGCCCAGCTCGACCTTCCCAAGTACACCATCATCAAGTTCTTTCAG2296    AACCAGCGGTACTATCTCAAGCACCACGGCAAACTGAAGGACAATTCC2344    GGTTTAGAGGTCGATGTGGCAGAATATAAAGAAGAGGAGCTGCTGAAG2392    GATTTGGAAGAGAGTGTCCAAGATAAAAATACTAACACCCTTTTTTCA2440    GTGAAACTAGAAGAAGAGCTGTCAGTGGAAGGAAACACAGACATTAAT2488    ACTGATTTGAAAGACTGAGATAAAAGTATTTGTTTCGTTCAACAGTGCCACTGGT2543    ATTTACTAACAAAATGAAAAGTCCACCTTGTCTTCTCTCAGAAAACCTTTGTTGTTCATT2603    GTTTGGCCAATGAACTTTCAAAAACTTGCACAAACAGAAAAGTTGGAAAAGGATAATACA2663    GACTGCACTAAATGTTTTCCTCTGTTTTACAAACTGCTTGGCAGCCCCAGGTGAAGCATC2723    AAGGATTGTTTGGTATTAAAATTTGTGTTCACGGGATGCACCAAAGTGTGTACCCCGTAA2783    GCATGAAACCAGTGTTTTTTGTTTTTTTTTTAGTTCTTATTCCGGAGCCTCAAACAAGCA2843    TTATACCTTCTGTGATTATGATTTCCTCTCCTATAATTATTTCTGTAGCACTCCACACTG2903    ATCTTTGGAAACTTGCCCCTTATTTAAAAAAAAAAAAAAAAAA2946    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 763 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    MetAspHisLeuAsnGluAlaThrGlnGlyLysGluHisSerGluMet    151015    SerAsnAsnValSerAspProLysGlyProProAlaLysIleAlaArg    202530    LeuGluGlnAsnGlySerProLeuGlyArgGlyArgLeuGlySerThr    354045    GlyAlaLysMetGlnGlyValProLeuLysHisSerGlyHisLeuMet    505560    LysThrAsnLeuArgLysGlyThrMetLeuProValPheCysValVal    65707580    GluHisTyrGluAsnAlaIleGluTyrAspCysLysGluGluHisAla    859095    GluPheValLeuValArgLysAspMetLeuPheAsnGlnLeuIleGlu    100105110    MetAlaLeuLeuSerLeuGlyTyrSerHisSerSerAlaAlaGlnAla    115120125    LysGlyLeuIleGlnValGlyLysTrpAsnProValProLeuSerTyr    130135140    ValThrAspAlaProAspAlaThrValAlaAspMetLeuGlnAspVal    145150155160    TyrHisValValThrLeuLysIleGlnLeuHisSerCysProLysLeu    165170175    GluAspLeuProProGluGlnTrpSerHisThrThrValArgAsnAla    180185190    LeuLysAspLeuLeuLysAspMetAsnGlnSerSerLeuAlaLysGlu    195200205    CysProLeuSerGlnSerMetIleSerSerIleValAsnSerThrTyr    210215220    TyrAlaAsnValSerAlaAlaLysCysGlnGluPheGlyArgTrpTyr    225230235240    LysHisPheLysLysThrLysAspMetMetValGluMetAspSerLeu    245250255    SerGluLeuSerGlnGlnGlyAlaAsnHisValAsnPheGlyGlnGln    260265270    ProValProGlyAsnThrAlaGluGlnProProSerProAlaGlnLeu    275280285    SerHisGlySerGlnProSerValArgThrProLeuProAsnLeuHis    290295300    ProGlyLeuValSerThrProIleSerProGlnLeuValAsnGlnGln    305310315320    LeuValMetAlaGlnLeuLeuAsnGlnGlnTyrAlaValAsnArgLeu    325330335    LeuAlaGlnGlnSerLeuAsnGlnGlnTyrLeuAsnHisProProPro    340345350    ValSerArgSerMetAsnLysProLeuGluGlnGlnValSerThrAsn    355360365    ThrGluValSerSerGluIleTyrGlnTrpValArgAspGluLeuLys    370375380    ArgAlaGlyIleSerGlnAlaValPheAlaArgValAlaPheAsnArg    385390395400    ThrGlnGlyLeuLeuSerGluIleLeuArgLysGluGluAspProLys    405410415    ThrAlaSerGlnSerLeuLeuValAsnLeuArgAlaMetGlnAsnPhe    420425430    LeuGlnLeuProGluAlaGluArgAspArgIleTyrGlnAspGluArg    435440445    GluArgSerLeuAsnAlaAlaSerAlaMetGlyProAlaProLeuIle    450455460    SerThrProProSerArgProProGlnValLysThrAlaThrIleAla    465470475480    ThrGluArgAsnGlyLysProGluAsnAsnThrMetAsnIleAsnAla    485490495    SerIleTyrAspGluIleGlnGlnGluMetLysArgAlaLysValSer    500505510    GlnAlaLeuPheAlaLysValAlaAlaThrLysSerGlnGlyTrpLeu    515520525    CysGluLeuLeuArgTrpLysGluAspProSerProGluAsnArgThr    530535540    LeuTrpGluAsnLeuSerMetIleArgArgPheLeuSerLeuProGln    545550555560    ProGluArgAspAlaIleTyrGluGlnGluSerAsnAlaValHisHis    565570575    HisGlyAspArgProProHisIleIleHisValProAlaGluGlnIle    580585590    GlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnAla    595600605    ProProProProGlnProGlnGlnGlnProGlnThrGlyProArgLeu    610615620    ProProArgGlnProThrValAlaSerProAlaGluSerAspGluGlu    625630635640    AsnArgGlnLysThrArgProArgThrLysIleSerValGluAlaLeu    645650655    GlyIleLeuGlnSerPheIleGlnAspValGlyLeuTyrProAspGlu    660665670    GluAlaIleGlnThrLeuSerAlaGlnLeuAspLeuProLysTyrThr    675680685    IleIleLysPhePheGlnAsnGlnArgTyrTyrLeuLysHisHisGly    690695700    LysLeuLysAspAsnSerGlyLeuGluValAspValAlaGluTyrLys    705710715720    GluGluGluLeuLeuLysAspLeuGluGluSerValGlnAspLysAsn    725730735    ThrAsnThrLeuPheSerValLysLeuGluGluGluLeuSerValGlu    740745750    GlyAsnThrAspIleAsnThrAspLeuLysAsp    755760763    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1960 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 112..886    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    CGCCCGCGCGCCCCGGGAGCCTACCCAGCACGCGCTCCGCGCCCACTGGTTCCCTCCAGC60    CGCCGCCGTCCAGCCGAGTCCCCACTCCGGAGTCGCCGCTGCCGCGGGGACATGGTC117    CTCTGCGTTCAGGGACCTCGTCCTTTGCTGGCTGTGGAGCGGACTGGG165    CAGCGGCCCCTGTGGGCCCCGTCCCTGGAACTGCCCAAGCCAGTCATG213    CAGCCCTTGCCTGCTGGGGCCTTCCTCGAGGAGGTGGCAGAGGGTACC261    CCAGCCCAGACAGAGAGTGAGCCAAAGGTGCTGGACCCAGAGGAGGAT309    CTGCTGTGCATAGCCAAGACCTTCTCCTACCTTCGGGAATCTGGCTGG357    TATTGGGGTTCCATTACGGCCAGCGAGGCCCGACAACACCTGCAGAAG405    ATGCCAGAAGGCACGTTCTTAGTACGTGACAGCACGCACCCCAGCTAC453    CTGTTCACGCTGTCAGTGAAAACCACTCGTGGCCCCACCAATGTACGC501    ATTGAGTATGCCGACTCCAGCTTCCGTCTGGACTCCAACTGCTTGTCC549    AGGCCACGCATCCTGGCCTTTCCGGATGTGGTCAGCCTTGTGCAGCAC597    TATGTGGCCTCCTGCACTGCTGATACCCGAAGCGACAGCCCCGATCCT645    GCTCCCACCCCGGCCCTGCCTATGCCTAAGGAGGATGCGCCTAGTGAC693    CCAGCACTGCCTGCTCCTCCACCAGCCACTGCTGTACACCTAAAACTG741    GTGCAGCCCTTTGTACGCAGAAGAAGTGCCCGCAGCCTGCAACACCTG789    TGCCGCCTTGTCATCAACCGTCTGGTGGCCGACGTGGACTGCCTGCCA837    CTGCCCCGGCGCATGGCCGACTACCTCCGACAGTACCCCTTCCAGCTCT886    GACTGTACGGGGCAATCTGCCCACCCTCACCCAGTCGCACCCTGGAGGGGACATCAGCCC946    CAGCTGGACTTGGGCCCCCACTGTCCCTCCTCCAGGCATCCTGGTGCCTGCATACCTCTG1006    GCAGCTGGCCCAGGAAGAGCCAGCAAGAGCAAGGCATGGGAGAGGGGAGGTGTCACACAA1066    CTTGGAGGTAAATGCCCCCAGGCCGCATGTGGCTTCATTATACTGAGCCATGTGTCAGAG1126    GATGGGGAGACAGGCAGGACCTTGTCTCACCTGTGGGCTGGGCCCAGACCTCCACTCGCT1186    TGCCTGCCCTGGCCACCTGAACTGTATGGGCACTCTCAGCCCTGGTTTTTCAATCCCCAG1246    GGTCGGGTAGGACCCCTACTGGCAGCCAGCCTCTGTTTCTGGGAGGATGACATGCAGAGG1306    AACTGAGATCGACAGTGACTAGTGACCCCTTGTTGAGGGGTAAGCCAGGCTAGGGGACTG1366    CACAATTATACACTCCTGAGCCCTGGTAGTCCAGAGACCCCAACTCTGCCCTGGCTTCTC1426    TGGTTCTTCCCTGTGGAAAGCCCATCCTGAGACATCTTGCTGGAACCAAGGCAATCCTGG1486    ATGTCCTGGTACTGACCCACCCGTCTGTGAATGTGTCCACTCTCTTCTGCCCCCAGCCAT1546    ATTTGGGGAGGATGGACAACTACAATAGGTAAGAAAATGCAGCCGGAGCCTCAGTCCCCA1606    GCAGAGCCTGTGTCTCACCCCCTCACAGGACAGAGCTGTATCTGCATAGAGCTGGTCTCA1666    CTGTGGCGCAGGCCCCGGGGGGAGTGCCTGTGCTGTCAGGAAGAGGGGGTGCTGGTTTGA1726    GGGCCACCACTGCAGTTCTGCTAGGTCTGCTTCCTGCCCAGGAAGGTGCCTGCACATGAG1786    AGGAGAGAAATACACGTCTGATAAGACTTCATGAAATAATAATTATAGCAAAGAACAGTT1846    TGGTGGTCTTTTCTCTTCCACTGATTTTTCTGTAATGACATTATACCTTTATTACCTCTT1906    TATTTTATTACCTCTATAATAAAATGATACCTTTCATGTAAAAAAAAAAAAAAA1960    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 258 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    MetValLeuCysValGlnGlyProArgProLeuLeuAlaValGluArg    151015    ThrGlyGlnArgProLeuTrpAlaProSerLeuGluLeuProLysPro    202530    ValMetGlnProLeuProAlaGlyAlaPheLeuGluGluValAlaGlu    354045    GlyThrProAlaGlnThrGluSerGluProLysValLeuAspProGlu    505560    GluAspLeuLeuCysIleAlaLysThrPheSerTyrLeuArgGluSer    65707580    GlyTrpTyrTrpGlySerIleThrAlaSerGluAlaArgGlnHisLeu    859095    GlnLysMetProGluGlyThrPheLeuValArgAspSerThrHisPro    100105110    SerTyrLeuPheThrLeuSerValLysThrThrArgGlyProThrAsn    115120125    ValArgIleGluTyrAlaAspSerSerPheArgLeuAspSerAsnCys    130135140    LeuSerArgProArgIleLeuAlaPheProAspValValSerLeuVal    145150155160    GlnHisTyrValAlaSerCysThrAlaAspThrArgSerAspSerPro    165170175    AspProAlaProThrProAlaLeuProMetProLysGluAspAlaPro    180185190    SerAspProAlaLeuProAlaProProProAlaThrAlaValHisLeu    195200205    LysLeuValGlnProPheValArgArgArgSerAlaArgSerLeuGln    210215220    HisLeuCysArgLeuValIleAsnArgLeuValAlaAspValAspCys    225230235240    LeuProLeuProArgArgMetAlaAspTyrLeuArgGlnTyrProPhe    245250255    GlnLeu    258    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1065 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 98..575    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    GTGGGTGCGCCGTGCTGAGCTCTGGCTGTCAGTGTGTTCGCCCGCGTCCCCTCCGCGCTC60    TCCGCTTGTGGATAACTAGCTGCTGGTTGATCGCACTATGACTCTGGAAGAAGTC115    CGCGGCCAGGACACAGTTCCGGAAAGCACAGCCAGGATGCAGGGTGCC163    GGGAAAGCGCTGCATGAGTTGCTGCTGTCGGCGCAGCGTCAGGGCTGC211    CTCACTGCCGGCGTCTACGAGTCAGCCAAAGTCTTGAACGTGGACCCC259    GACAATGTGACCTTCTGTGTGCTGGCTGCGGGTGAGGAGGACGAGGGC307    GACATCGCGCTGCAGATCCATTTTACGCTGATCCAGGCTTTCTGCTGC355    GAGAACGACATCGACATAGTGCGCGTGGGCGATGTGCAGCGGCTGGCG403    GCTATCGTGGGCGCCGGCGAGGAGGCGGGTGCGCCGGGCGACCTGCAC451    TGCATCCTCATTTCGAACCCCAACGAGGACGCCTGGAAGGATCCCGCC499    TTGGAGAAGCTCAGCCTGTTTTGCGAGGAGAGCCGCAGCGTTAACGAC547    TGGGTGCCCAGCATCACCCTCCCCGAGTGACAGCCCGGCGGGGACCTT595    GGTCTGATCGACGTGGTGACGCCCCGGGGCGCCTAGAGCGCGGCTGGCTCTGTGGAGGGG655    CCCTCCGAGGGTGCCCGAGTGCGGCGTGGAGACTGGCAGGCGGGGGGGGCGCCTGGAGAG715    CGAGGAGGCGCGGCCTCCCGAGGAGGGGCCCGGTGGCGGCAGGGCCAGGCTGGTCCGAGC775    TGAGGACTCTGCAAGTGTCTGGAGCGGCTGCTCGCCCAGGAAGGCCTAGGCTAGGACGTT835    GGCCTCAGGGCCAGGAAGGACAGACTGGCCGGGCAGGCGTGACTCAGCAGCCTGCGCTCG895    GCAGGAAGGAGCGGCGCCCTGGACTTGGTACAGTTTCAGGAGCGTGAAGGACTTAACCGA955    CTGCCGCTGCTTTTTCAAAACGGATCCGGGCAATGCTTCGTTTTCTAAAGGATGCTGCTG1015    TTGAGCTTTGAATTTTACAATAAACTTTTTGAAACAAAAAAAAAAAAAAA1065    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 159 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    MetThrLeuGluGluValArgGlyGlnAspThrValProGluSerThr    151015    AlaArgMetGlnGlyAlaGlyLysAlaLeuHisGluLeuLeuLeuSer    202530    AlaGlnArgGlnGlyCysLeuThrAlaGlyValTyrGluSerAlaLys    354045    ValLeuAsnValAspProAspAsnValThrPheCysValLeuAlaAla    505560    GlyGluGluAspGluGlyAspIleAlaLeuGlnIleHisPheThrLeu    65707580    IleGlnAlaPheCysCysGluAsnAspIleAspIleValArgValGly    859095    AspValGlnArgLeuAlaAlaIleValGlyAlaGlyGluGluAlaGly    100105110    AlaProGlyAspLeuHisCysIleLeuIleSerAsnProAsnGluAsp    115120125    AlaTrpLysAspProAlaLeuGluLysLeuSerLeuPheCysGluGlu    130135140    SerArgSerValAsnAspTrpValProSerIleThrLeuProGlu    145150155    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 2980 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 240..1475    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    CACACCGCCAGTCTGTGCGCTGAGTCGGAGCCAGAGGCCGCGGGGACACCGGGCCATGCA60    CGCCCCCAACTGAAGCTGCATCTCAAAGCCGAAGATTCCAGCAGCCCAGGGGATTTCAAA120    GAGCTCAGACTCAGAGGAACATCTGCGGAGAGACCCCCGAAGCCCTCTCCAGGGCAGTCC180    TCATCCAGACGCTCCGTTAGTGCAGACAGGAGCGCGCAGTGGCCCCGGCTCGCCGCGCC239    ATGGAGCGGATCCCCAGCGCGCAACCACCCCCCGCCTGCCTGCCCAAA287    GCACCGGGACTGGAGCACCGAGACCTACCAGGGATGTACCCTGCCCAC335    ATGTACCAAGTGTACAAGTCAAGACGGGGAATAAAGCGGAGCGAGGAC383    AGCAAGGAGACCTACAAATTGCCGCACCGGCTCTTCGAGAAAAAGAGA431    CGTGACCGGATTAACGAGTGCATCGCCCAGCTGAAGGATCTCCTACCC479    GAACATCTCAAACTTACAACTTTGGGTCACTTGGAAAAAGCAGTGGTT527    CTTGAACTTACCTTGAAGCATGTGAAAGCACTAACAAACCTAATTGAT575    CAGCAGCAGCAGAAAATCATTGCCCTGCAGAGTGGTTTACAAGCTGGT623    GAGCTGTCAGGGAGAAATGTCGAAACAGGTCAAGAGATGTTCTGCTCA671    GGTTTCCAGACATGTGCCCGGGAGGTGCTTCAGTATCTGGCCAAGCAC719    GAGAACACTCGGGACCTGAAGTCTTCGCAGCTTGTCACCCACCTCCAC767    CGGGTGGTCTCGGAGCTGCTGCAGGGTGGTACCTCCAGGAAGCCATCA815    GACCCAGCTCCCAAAGTGATGGACTTCAAGGAAAAACCCAGCTCTCCG863    GCCAAAGGTTCGGAAGGTCCTGGGAAAAACTGCGTGCCAGTCATCCAG911    CGGACTTTCGCTCACTCGAGTGGGGAGCAGAGCGGCAGCGACACGGAC959    ACAGACAGTGGCTATGGAGGAGATTCGGAGAAGGGCGACTTGCGCAGT1007    GAGCAGCCGTGCTTCAAAAGTGACCACGGACGCAGGTTCACGATGGGA1055    GAAAGGATCGGCGCAATTAAGCAAGAGTCCGAAGAACCCCCCACAAAA1103    AAGAACCGGATGCAGCTTTCGGATGATGAAGGCCATTTCACTAGCAGT1151    GACCTGATCAGCTCCCCGTTCCTGGGCCCACACCCACACCAGCCTCCT1199    TTCTGCCTGCCCTTCTACCTGATCCCACCTTCAGCGACTGCCTACCTG1247    CCCATGCTGGAGAAGTGCTGGTATCCCACCTCAGTGCCAGTGCTATAC1295    CCAGGCCTCAACGCCTCTGCCGCAGCCCTCTCTAGCTTCATGAACCCA1343    GACAAGATCTCGGCTCCCTTGCTCATGCCCCAGAGACTCCCTTCTCCC1391    TTGCCAGCTCATCCGTCCGTCGACTCTTCTGTCTTGCTCCAAGCTCTG1439    AAGCCAATCCCCCCTTTAAACTTAGAAACCAAAGACTAAACTCTCTA1486    GGGGATCCTGCTGCTTNGCTTTCCTNCCTCGCTACTTCCTAAAAAGCAACCNNAAAGNTT1546    TNGTGAATGCTGNNAGANTGTTGCATTGTGTATACTGAGATAATCTGAGGCATGGAGAGC1606    AGANNCAGGGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTATGTGCGTGTGCGTGCACA1666    TGTGTGCCTGCGTGTTGGTATAGGACTTTANNGCTCCTTNNGGCATAGGGAAGTCACGAA1726    GGATTGCTNGACATCAGGAGACTNGGGGGGGATTGTAGCAGACGTCTGGGCTTNNCCCCA1786    CCCAGAGAATAGCCCCCNNCNANACANATCAGCTGGATTTACAAAAGCTTCAAAGTCTTG1846    GTCTGTGAGTCACTCTTCAGTTTGGGAGCTGGGTCTGTGGCTTTGATCAGAAGGTACTTT1906    CAAAAGAGGGCTTTCCAGGGCTCAGCTCCCAACCAGCTGTTAGGACCCCACCCTTTTGCC1966    TTTATTGTCGACGTGACTCACCAGACGTCGGGGAGAGAGAGCAGTCAGACCGAGCTTTTC2026    TGCTAACATGGGGAGGGTAGCAGACACTGGCATAGCACGGTAGTGGTTTGGGGGAGGGTT2086    TCCGCAGGTCTGCTCCCCACCCCTGCCTCGGAAGAATAAAGAGAATGTAGTTCCCTACTC2146    AGGCTTTCGTAGTGATTAGCTTACTAAGGAACTGAAAATGGGCCCCTTGTACAAGCTGAG2206    CTGCCCCGGAGGGAGGGAGGAGTTCCCTGGGCTTCTGGCACCTGTTTCTAGGCCTAACCA2266    TTAGTACTTACTGTGCAGGGAACCAAACCAAGGTCTGAGAAATGCGGACANCCCGAGCGA2326    GCACCCCAAAGTGCACAAAGCTGAGTAAAAAGCTGCCCCCTTCAAACAGAACTAGACTCA2386    GTTTTCAATTCCATCCTAAAACTCCTTTTAACCAAGCTTAGCTTCTCAAAGGGCTAACCA2446    AGCCTTGGAACCGCCAGATCCTTTCTGTAGGCTAATTCCTCTTGGCCAACGGCATATGGA2506    GTGTCCTTATTGCTAAAAAGGATTCCGNCTCCTTCAAAGAAGTTTTATTTTTGGTCCAGA2566    GTACTTGTTTTCCCGATGTGTCCAGCCAGCTCCGCAGCAGCTTTTCAAAATGCACTATGC2626    CTGATTGCTGATCGTGTTTTAACTTTTTCTTTTCCTGTTTTTATTTTGGTATTAAGTCGC2686    TGGCTTTATTTGTAAAGCTGTTATAAATATATATTATATNAANTATATTAAAAAGGAAAN2746    TGTTNCAGATGTTTATTTGTATAATTACTTGATTCACANAGNGAGAAAAANTGANTGTAT2806    TCCTGTNTTNGAAGAGAAGANNAATTTTTTTTTTCTCTAGGGAGAGGTACAGNGTTNNTN2866    TTTTGGGGCCTNCCNGAAGGGGTAAANNNGAAAATNTTTCTATNTATGAGTAAATGTTAA2926    GTAGTTGTNTNAAAATACTNAATAAAATAATTCTCTCCCTGTGGNNGAGANAAC2980    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 412 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    MetGluArgIleProSerAlaGlnProProProAlaCysLeuProLys    151015    AlaProGlyLeuGluHisArgAspLeuProGlyMetTyrProAlaHis    202530    MetTyrGlnValTyrLysSerArgArgGlyIleLysArgSerGluAsp    354045    SerLysGluThrTyrLysLeuProHisArgLeuPheGluLysLysArg    505560    ArgAspArgIleAsnGluCysIleAlaGlnLeuLysAspLeuLeuPro    65707580    GluHisLeuLysLeuThrThrLeuGlyHisLeuGluLysAlaValVal    859095    LeuGluLeuThrLeuLysHisValLysAlaLeuThrAsnLeuIleAsp    100105110    GlnGlnGlnGlnLysIleIleAlaLeuGlnSerGlyLeuGlnAlaGly    115120125    GluLeuSerGlyArgAsnValGluThrGlyGlnGluMetPheCysSer    130135140    GlyPheGlnThrCysAlaArgGluValLeuGlnTyrLeuAlaLysHis    145150155160    GluAsnThrArgAspLeuLysSerSerGlnLeuValThrHisLeuHis    165170175    ArgValValSerGluLeuLeuGlnGlyGlyThrSerArgLysProSer    180185190    AspProAlaProLysValMetAspPheLysGluLysProSerSerPro    195200205    AlaLysGlySerGluGlyProGlyLysAsnCysValProValIleGln    210215220    ArgThrPheAlaHisSerSerGlyGluGlnSerGlySerAspThrAsp    225230235240    ThrAspSerGlyTyrGlyGlyAspSerGluLysGlyAspLeuArgSer    245250255    GluGlnProCysPheLysSerAspHisGlyArgArgPheThrMetGly    260265270    GluArgIleGlyAlaIleLysGlnGluSerGluGluProProThrLys    275280285    LysAsnArgMetGlnLeuSerAspAspGluGlyHisPheThrSerSer    290295300    AspLeuIleSerSerProPheLeuGlyProHisProHisGlnProPro    305310315320    PheCysLeuProPheTyrLeuIleProProSerAlaThrAlaTyrLeu    325330335    ProMetLeuGluLysCysTrpTyrProThrSerValProValLeuTyr    340345350    ProGlyLeuAsnAlaSerAlaAlaAlaLeuSerSerPheMetAsnPro    355360365    AspLysIleSerAlaProLeuLeuMetProGlnArgLeuProSerPro    370375380    LeuProAlaHisProSerValAspSerSerValLeuLeuGlnAlaLeu    385390395400    LysProIleProProLeuAsnLeuGluThrLysAsp    405410412    (2) INFORMATION FOR SEQ ID NO:15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    GGGGTCTACCAGGGATGTAC20    (2) INFORMATION FOR SEQ ID NO:16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    GTAAACCACTCTGCAGGGCAATGA24    (2) INFORMATION FOR SEQ ID NO:17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 28 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    GATCGTAGTCACGCAGGTGGGATCCCTA28    (2) INFORMATION FOR SEQ ID NO:18:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 28 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    GATCTAGGGATCCCACCTGCGTGACTAC28    (2) INFORMATION FOR SEQ ID NO:19:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 28 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    GATCGGTGTAGGCCACGTGACCGGGTGT28    (2) INFORMATION FOR SEQ ID NO:20:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 28 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    GATCACACCCGGTCACGTGGCCTACACC28    (2) INFORMATION FOR SEQ ID NO:21:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:    GATCGGCAGCCGGCACGCGACAGGGCC27    (2) INFORMATION FOR SEQ ID NO:22:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:    GATCGGCCCTGTCGCGTGCCGGCTGCC27    (2) INFORMATION FOR SEQ ID NO:23:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:    GATCACGCCACGAGCCACAAGGATTG26    (2) INFORMATION FOR SEQ ID NO:24:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:    GATCCAATCCTTGTGGCTCGTGGCGT26    (2) INFORMATION FOR SEQ ID NO:25:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 2297 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:    GCGCCGCATCCTGGAGGTTGGGATGCTCTTGTCCAAAATCAACTCGCTTG50    CCCACCTGCGCGCCCGCGCCTGCAACGACCTGCACGCCACCAAGCTGGCG100    CCGGGCAAGGAGAAGGAGCCCCTGGAGTCGCAGTACCAGGTGGGCCCGCT150    ACTGGGCAGCGGCGGCTTCGGCTCGGTCTACTCAGGCATCCGCGTCTCCG200    ACAACTTGCCGGTGGCCATCAAACACGTGGAGAAGGACCGGATTTCCGAC250    TGGGGAGAGCTGCCTAATGGCACTCGAGTGCCCATGGAAGTGGTCCTGCT300    GAAGAAGGTGAGCTCGGGTTTCTCCGGCGTCATTAGGCTCCTGGACTGGT350    TCGAGAGGCCCGACAGTTTCGTCCTGATCCTGGAGAGGCCCGAGCCGGTG400    CAAGATCTCTTCGACTTCATCACGGAAAGGGGAGCCCTGCAAGAGGAGCT450    GGCCCGCAGCTTCTTCTGGCAGGTGCTGGAGGCCGTGCGGCACTGCCACA500    ACTGCGGGGTGCTCCACCGCGACATCAAGGACGAAAACATCCTTATCGAC550    CTCAATCGCGGCGAGCTCAAGCTCATCGACTTCGGGTCGGGGGCGCTGCT600    CAAGGACACCGTCTACACGGACTTCGATGGGACCCGAGTGTATAGCCCTC650    CAGAGTGGATCCGCTACCATCGCTACCATGGCAGGTCGGCGGCAGTCTGG700    TCCCTGGGGATCCTGCTGTATGATATGGTGTGTGGAGATATTCCTTTCGA750    GCATGACGAAGAGATCATCAGGGGCCAGGTTTTCTTCAGGCAGAGGGTCT800    CTTCAGAATGTCAGCATCTCATTAGATGGTGCTTGGCCCTGAGACCATCA850    GATAGGCCAACCTTCGAAGAAATCCAGAACCATCCATGGATGCAAGATGT900    TCTCCTGCCCCAGGAAACTGCTGAGATCCACCTCCACAGCCTGTCGCCGG950    GGCCCAGCAAATAGCAGCCTTTCTGGCAGGTCCTCCCCTCTCTTGTCAGA1000    TGCCCAGGAGGGAAGCTTCTGTCTCCAGCTTTCCCGAGTACCAGTGACAC1050    GTCTCGCCAAGCAGGACAGTGCTTGATACAGGAACAACATTTACAACTCA1100    TTCCAGATCCCAGGCCCCTGGAGGCTGCCTCCCAACAGTGGGGAAGAGTG1150    ACTCTCCAGGGGTCCTAGGCCTCAACTCCTCCCATAGATACTCTCTTCTT1200    CTCATAGGTGTCCAGCATTGCTGGACTCTGAAATATCCCGGGGGTGGGGG1250    GTGGGGGTGGGTCAGAACCCTGCCATGGAACTGTTTCCTTCATCATGAGT1300    TCTGCTGAATGCCGCGATGGGTCAGGTAGGGGGGAAACAGGTTGGGATGG1350    GATAGGACTAGCACCATTTTAAGTCCCTGTCACCTCTTCCGACTCTTTCT1400    GAGTGCCTTCTGTGGGGACTCCGGCTGTGCTGGGAGAAATACTTGAACTT1450    GCCTCTTTTACCTGCTGCTTCTCCAAAAATCTGCCTGGGTTTTGTTCCCT1500    ATTTTTCTCTCCTGTCCTCCCTCACCCCCTCCTTCATATGAAAGGTGCCA1550    TGGAAGAGGCTACAGGGCCAAACGCTGAGCCACCTGCCCTTTTTTCTCCT1600    CCTTTAGTAAAACTCCGAGTGAACTGGTCTTCCTTTTTGGTTTTTACTTA1650    ACTGTTTCAAAGCCAAGACCTCACACACACAAAAAATGCACAAACAATGC1700    AATCAACAGAAAAGCTGTAAATGTGTGTACAGTTGGCATGGTAGTATACA1750    AAAAGATTGTAGTGGATCTAATTTTTAAGAAATTTTGCCTTTAAGTTATT1800    TTACCTGTTTTTGTTTCTTGTTTTGAAAGATGCGCATTCTAACCTGGAGG1850    TCAATGTTATGTATTTATTTATTTATTTATTTGGTTCCCTTCCTANNNNN1900    NNNNNNGCTGCTGCCCTAGTTTTCTTTCCTCCTTTCCTCCTCTGACTTGG1950    GGACCTTTTGGGGGAGGGCTGCGACGCTTGCTCTGTTTGTGGGGTGACGG2000    GACTCAGGCGGGACAGTGCTGCAGCTCCCTGGCTTCTGTGGGGCCCCTCA2050    CCTACTTACCCAGGTGGGTCCCGGCTCTGTGGGTGATGGGGAGGGGCATT2100    GCTGACTGTGTATATAGGATAATTATGAAAAGCAGTTCTGGATGGTGTGC2150    CTTCCAGATCCTCTCTGGGGCTGTGTTTTGAGCAGCAGGTAGCCTGCTGG2200    TTTTATCTGAGTGAAATACTGTACAGGGGAATAAAAGAGATCTTATTTTT2250    TTTTTTATACTTGGCGTTTTTTGAATAAAAACCTTTTGTCTTAAAAC2297    (2) INFORMATION FOR SEQ ID NO:26:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 313 amino acids    (B) TYPE:AMINO    (B) TYPE:AMINO    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: Not Relevant    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:    MetLeuLeuSerLysIleAsnSerLeuAla    1510    HisLeuArgAlaArgAlaCysAsnAspLeuHisAlaThrLysLeuAla    152025    ProGlyLysGluLysGluProLeuGluSerGlnTyrGlnValGlyPro    303540    LeuLeuGlySerGlyGlyPheGlySerValTyrSerGlyIleArgVal    455055    SerAspAsnLeuProValAlaIleLysHisValGluLysAspArgIle    606570    SerAspTrpGlyGluLeuProAsnGlyThrArgValProMetGluVal    75808590    ValLeuLeuLysLysValSerSerGlyPheSerGlyValIleArgLeu    95100105    LeuAspTrpPheGluArgProAspSerPheValLeuIleLeuGluArg    110115120    ProGluProValGlnAspLeuPheAspPheIleThrGluArgGlyAla    125130135    LeuGlnGluGluLeuAlaArgSerPhePheTrpGlnValLeuGluAla    140145150    ValArgHisCysHisAsnCysGlyValLeuHisArgAspIleLysAsp    155160165170    GluAsnIleLeuIleAspLeuAsnArgGlyGluLeuLysLeuIleAsp    175180185    PheGlySerGlyAlaLeuLeuLysAspThrValTyrThrAspPheAsp    190195200    GlyThrArgValTyrSerProProGluTrpIleArgTyrHisArgTyr    205210215    HisGlyArgSerAlaAlaValTrpSerLeuGlyIleLeuLeuTyrAsp    220225230    MetValCysGlyAspIleProPheGluHisAspGluGluIleIleArg    235240245250    GlyGlnValPhePheArgGlnArgValSerSerGluCysGlnHisLeu    255260265    IleArgTrpCysLeuAlaLeuArgProSerAspArgProThrPheGlu    270275280    GluIleGlnAsnHisProTrpMetGlnAspValLeuLeuProGlnGlu    285290295    ThrAlaGluIleHisLeuHisSerLeuSerProGlyProSerLys    300305310313    (2) INFORMATION FOR SEQ ID NO:27:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 606 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:    ATG3    TGCCGCACCCTGGCCGCCTTCCCCACCACCTGCCTGGAGAGAGCCAAA51    GAGTTCAAGACACGTCTGGGGATCTTTCTTCACAAATCAGAGCTGGGC99    TGCGATACTGGGAGTACTGGCAAGTTCGAGTGGGGCAGTAAACACAGC147    AAAGAGAATAGAAACTTCTCAGAAGATGTGCTGGGGTGGAGAGAGTCG195    TTCGACCTGCTGCTGAGCAGTAAAAATGGAGTGGCTGCCTTCCACGCT243    TTCCTGAAGACAGAGTTCAGTGAGGAGAACCTGGAGTTCTGGCTGGCC291    TGTGAGGAGTTCAAGAAGATCCGATCAGCTACCAAGCTGGCCTCCAGG339    GCACACCAGATCTTTGAGGAGTTCATTTGCAGTGAGGCCCCTAAAGAG387    GTCAACATTGACCATGAGACCCGCGAGCTGACGAGGATGAACCTGCAG435    ACTGCCACAGCCACATGCTTTGATGCGGCTCAGGGGAAGACACGTACC483    CTGATGGAGAAGGACTCCTACCCACGCTTCCTGAAGTCGCCTGCTTAC531    CGGGACCTGGCTGCCCAAGCCTCAGCCGCCTCTGCCACTCTGTCCAGC579    TGCAGCCTGGACCAGCCCTCACACACC606    (2) INFORMATION FOR SEQ ID NO:28:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 180 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:    ATGGGG6    ACGTTAAGCATGCAGCAACTACAGTCATTTGTTCTCAGAGGTCTGGAC54    CAAAGAGAAACAAGAAAAGCTGGAGTCACACTACCAAAGGCCGAAGCT102    GAGCAACAGAGCTCTGGAGTCAGCTGCCTGGGTTCAGCATGCAGCGCT150    GCCGTGGACGATCTGTCTCTCTTGCATATA180    (2) INFORMATION FOR SEQ ID NO: 29:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1074 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:    ATGGGAAAT9    GCCTCCAATGACTCCCAGTCTGAGGACTGCGAGACGCGACAGTGGTTT57    CCCCCAGGCGAAAGCCCAGCCATCAGTTCCGTCATGTTCTCGGCCGGG105    GTGCTGGGGAACCTCATAGAACTGGCGCTGCTGGCGCGCCGCTGGCAG153    GGGGACGTGGGGTGCAGCGCCGGCCGTAGGAGCTCCCTCTCCTTGTTC201    CACGTGCTGGTGACCGAGCTGGTGTTCACCGACCTGCTCGGGACCTGC249    CTCATCAGCCCAGTGGTACTGGCTTCGTACGCGCGGAACCAGACCCTG297    GTGGCACTGGCGCCCGAGAGCCGCGCGTCCACCTACTTCGCTTTCGCC345    ATGACCTTCTTCAGCCTGGCCACGATGCTCATGCTCTTCACCATGGCC393    CTGGAGCGCTACCTCTCGATCGGGCACCCCTACTTCTACCAGCGCCGC441    GTCTCGCGCTCCGGGGGCCTGGCCGTGCTGCCTGTCATCTATGCAGTC489    TCCCTGCTCTTCTGCTCACTGCCGCTGCTGGACTATGGGCAGTACGTC537    CAGTACTGCCCCGGGACCTGGTGCTTCATCCGGCACGGGCGGACCGCT585    TACCTGCAGCTGTACGCCACCCTGCTGCTGCTTCTCATTGTCTCGGTG633    CTCGCCTGCAACTTCAGTGTCATTCTCAACCTCATCCGCATGCACCGC681    CGAAGCCGGAGAAGCCGCTGCGGACCTTCCCTGGGCAGTGGCCGGGGC729    GGCCCCGGGGCCCGCAGGAGAGGGGAAAGGGTGTCCATGGCGGAGGAG777    ACGGACCACCTCATTCTCCTGGCTATCATGACCATCACCTTCGCCGTC825    TGCTCCTTGCCTTTCACGATTTTTGCATATATGAATGAAACCTCTTCC873    CGAAAGGAAAAATGGGACCTCCAAGCTCTTAGGTTTTTATCAATTAAT921    TCAATAATTGACCCTTGGGTCTTTGCCATCCTTAGGCCTCCTGTTCTG969    AGACTAATGCGTTCAGTCCTCTGTTGTCGGATTTCATTAAGAACACAA1017    GATGCAACACAAACTTCCTGTTCTACACAGTCAGATGCCAGTAAACAG1065    GCTGACCTT1074    (2) INFORMATION FOR SEQ ID NO: 30:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 2289 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:    ATGGATCATTTGAACGAG18    GCAACTCAGGGGAAAGAACATTCAGAAATGTCTAACAATGTGAGTGAT66    CCGAAGGGTCCACCAGCCAAGATTGCCCGCCTGGAGCAGAACGGGAGC114    CCGCTAGGAAGAGGAAGGCTTGGGAGTACAGGTGCAAAAATGCAGGGA162    GTGCCTTTAAAACACTCGGGCCATCTGATGAAAACCAACCTTAGGAAA210    GGAACCATGCTGCCAGTTTTCTGTGTGGTGGAACATTATGAAAACGCC258    ATTGAATATGATTGCAAGGAGGAGCATGCAGAATTTGTGCTGGTGAGA306    AAGGATATGCTTTTCAACCAGCTGATCGAAATGGCATTGCTGTCTCTA354    GGTTATTCACATAGCTCTGCTGCCCAGGCCAAAGGGCTAATCCAGGTT402    GGAAAGTGGAATCCAGTTCCACTGTCTTACGTGACAGATGCCCCTGAT450    GCTACAGTAGCAGATATGCTTCAAGATGTGTATCATGTGGTCACATTG498    AAAATTCAGTTACACAGTTGCCCCAAACTAGAAGACTTGCCTCCCGAA546    CAATGGTCGCACACCACAGTGAGGAATGCTCTGAAGGACTTACTGAAA594    GATATGAATCAGAGTTCATTGGCCAAGGAGTGCCCCCTTTCACAGAGT642    ATGATTTCTTCCATTGTGAACAGTACTTACTATGCAAATGTCTCAGCA690    GCAAAATGTCAAGAATTTGGAAGGTGGTACAAACATTTCAAGAAGACA738    AAAGATATGATGGTTGAAATGGATAGTCTTTCTGAGCTATCCCAGCAA786    GGCGCCAATCATGTCAATTTTGGCCAGCAACCAGTTCCAGGGAACACA834    GCCGAGCAGCCTCCATCCCCTGCGCAGCTCTCCCATGGCAGCCAGCCC882    TCTGTCCGGACACCTCTTCCAAACCTGCACCCTGGGCTCGTATCAACA930    CCTATCAGTCCTCAATTGGTCAACCAGCAGCTGGTGATGGCTCAGCTG978    CTGAACCAGCAGTATGCAGTGAATAGACTTTTAGCCCAGCAGTCCTTA1026    AACCAACAATACTTGAACCACCCTCCCCCTGTCAGTAGATCTATGAAT1074    AAGCCTTTGGAGCAACAGGTTTCGACCAACACAGAGGTGTCTTCCGAA1122    ATCTACCAGTGGGTACGCGATGAACTGAAACGAGCAGGAATCTCCCAG1170    GCGGTATTTGCACGTGTGGCTTTTAACAGAACTCAGGGCTTGCTTTCA1218    GAAATCCTCCGAAAGGAAGAGGACCCCAAGACTGCATCCCAGTCTTTG1266    CTGGTAAACCTTCGGGCTATGCAGAATTTCTTGCAGTTACCGGAAGCT1314    GAAAGAGACCGAATATACCAGGACGAAAGGGAAAGGAGCTTGAATGCT1362    GCCTCGGCCATGGGTCCTGCCCCCCTCATCAGCACACCACCCAGCCGT1410    CCTCCCCAGGTGAAAACAGCTACTATTGCCACTGAAAGGAATGGGAAA1458    CCAGAGAACAATACCATGAACATTAATGCTTCCATTTATGATGAGATT1506    CAGCAGGAAATGAAGCGTGCTAAAGTGTCTCAAGCACTGTTTGCAAAG1554    GTTGCAGCAACCAAAAGCCAGGGATGGTTGTGCGAGCTGTTACGCTGG1602    AAAGAAGATCCTTCTCCAGAAAACAGAACCCTGTGGGAGAACCTCTCC1650    ATGATCCGAAGGTTCCTCAGTCTTCCTCAGCCAGAACGTGATGCCATT1698    TATGAACAGGAGAGCAACGCGGTGCATCACCATGGCGACAGGCCGCCC1746    CACATTATCCATGTTCCAGCAGAGCAGATTCAGCAACAGCAGCAGCAA1794    CAGCAACAGCAGCAGCAGCAGCAGCAGGCACCGCCGCCTCCACAGCCA1842    CAGCAGCAGCCACAGACAGGCCCTCGGCTCCCCCCACGGCAACCCACG1890    GTGGCCTCTCCAGCAGAGTCAGATGAGGAAAACCGACAGAAGACCCGG1938    CCACGAACAAAAATTTCAGTGGAAGCCTTGGGAATCCTCCAGAGTTTC1986    ATACAAGACGTGGGCCTGTACCCTGACGAAGAGGCCATCCAGACTCTG2034    TCTGCCCAGCTCGACCTTCCCAAGTACACCATCATCAAGTTCTTTCAG2082    AACCAGCGGTACTATCTCAAGCACCACGGCAAACTGAAGGACAATTCC2130    GGTTTAGAGGTCGATGTGGCAGAATATAAAGAAGAGGAGCTGCTGAAG2178    GATTTGGAAGAGAGTGTCCAAGATAAAAATACTAACACCCTTTTTTCA2226    GTGAAACTAGAAGAAGAGCTGTCAGTGGAAGGAAACACAGACATTAAT2274    ACTGATTTGAAAGAC2289    (2) INFORMATION FOR SEQ ID NO: 31:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 477 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:    ATGACTCTGGAAGAAGTC18    CGCGGCCAGGACACAGTTCCGGAAAGCACAGCCAGGATGCAGGGTGCC66    GGGAAAGCGCTGCATGAGTTGCTGCTGTCGGCGCAGCGTCAGGGCTGC114    CTCACTGCCGGCGTCTACGAGTCAGCCAAAGTCTTGAACGTGGACCCC162    GACAATGTGACCTTCTGTGTGCTGGCTGCGGGTGAGGAGGACGAGGGC210    GACATCGCGCTGCAGATCCATTTTACGCTGATCCAGGCTTTCTGCTGC258    GAGAACGACATCGACATAGTGCGCGTGGGCGATGTGCAGCGGCTGGCG306    GCTATCGTGGGCGCCGGCGAGGAGGCGGGTGCGCCGGGCGACCTGCAC354    TGCATCCTCATTTCGAACCCCAACGAGGACGCCTGGAAGGATCCCGCC402    TTGGAGAAGCTCAGCCTGTTTTGCGAGGAGAGCCGCAGCGTTAACGAC450    TGGGTGCCCAGCATCACCCTCCCCGAG477    (2) INFORMATION FOR SEQ ID NO: 32:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1236 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:    ATGGAGCGGATCCCCAGCGCGCAACCACCCCCCGCCTGCCTGCCCAAA48    GCACCGGGACTGGAGCACCGAGACCTACCAGGGATGTACCCTGCCCAC96    ATGTACCAAGTGTACAAGTCAAGACGGGGAATAAAGCGGAGCGAGGAC144    AGCAAGGAGACCTACAAATTGCCGCACCGGCTCTTCGAGAAAAAGAGA192    CGTGACCGGATTAACGAGTGCATCGCCCAGCTGAAGGATCTCCTACCC240    GAACATCTCAAACTTACAACTTTGGGTCACTTGGAAAAAGCAGTGGTT288    CTTGAACTTACCTTGAAGCATGTGAAAGCACTAACAAACCTAATTGAT336    CAGCAGCAGCAGAAAATCATTGCCCTGCAGAGTGGTTTACAAGCTGGT384    GAGCTGTCAGGGAGAAATGTCGAAACAGGTCAAGAGATGTTCTGCTCA432    GGTTTCCAGACATGTGCCCGGGAGGTGCTTCAGTATCTGGCCAAGCAC480    GAGAACACTCGGGACCTGAAGTCTTCGCAGCTTGTCACCCACCTCCAC528    CGGGTGGTCTCGGAGCTGCTGCAGGGTGGTACCTCCAGGAAGCCATCA576    GACCCAGCTCCCAAAGTGATGGACTTCAAGGAAAAACCCAGCTCTCCG624    GCCAAAGGTTCGGAAGGTCCTGGGAAAAACTGCGTGCCAGTCATCCAG672    CGGACTTTCGCTCACTCGAGTGGGGAGCAGAGCGGCAGCGACACGGAC720    ACAGACAGTGGCTATGGAGGAGATTCGGAGAAGGGCGACTTGCGCAGT768    GAGCAGCCGTGCTTCAAAAGTGACCACGGACGCAGGTTCACGATGGGA816    GAAAGGATCGGCGCAATTAAGCAAGAGTCCGAAGAACCCCCCACAAAA864    AAGAACCGGATGCAGCTTTCGGATGATGAAGGCCATTTCACTAGCAGT912    GACCTGATCAGCTCCCCGTTCCTGGGCCCACACCCACACCAGCCTCCT960    TTCTGCCTGCCCTTCTACCTGATCCCACCTTCAGCGACTGCCTACCTG1008    CCCATGCTGGAGAAGTGCTGGTATCCCACCTCAGTGCCAGTGCTATAC1056    CCAGGCCTCAACGCCTCTGCCGCAGCCCTCTCTAGCTTCATGAACCCA1104    GACAAGATCTCGGCTCCCTTGCTCATGCCCCAGAGACTCCCTTCTCCC1152    TTGCCAGCTCATCCGTCCGTCGACTCTTCTGTCTTGCTCCAAGCTCTG1200    AAGCCAATCCCCCCTTTAAACTTAGAAACCAAAGAC1236    (2) INFORMATION FOR SEQ ID NO: 33:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 774 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:    ATGGTC6    CTCTGCGTTCAGGGACCTCGTCCTTTGCTGGCTGTGGAGCGGACTGGG54    CAGCGGCCCCTGTGGGCCCCGTCCCTGGAACTGCCCAAGCCAGTCATG102    CAGCCCTTGCCTGCTGGGGCCTTCCTCGAGGAGGTGGCAGAGGGTACC150    CCAGCCCAGACAGAGAGTGAGCCAAAGGTGCTGGACCCAGAGGAGGAT198    CTGCTGTGCATAGCCAAGACCTTCTCCTACCTTCGGGAATCTGGCTGG246    TATTGGGGTTCCATTACGGCCAGCGAGGCCCGACAACACCTGCAGAAG294    ATGCCAGAAGGCACGTTCTTAGTACGTGACAGCACGCACCCCAGCTAC342    CTGTTCACGCTGTCAGTGAAAACCACTCGTGGCCCCACCAATGTACGC390    ATTGAGTATGCCGACTCCAGCTTCCGTCTGGACTCCAACTGCTTGTCC438    AGGCCACGCATCCTGGCCTTTCCGGATGTGGTCAGCCTTGTGCAGCAC486    TATGTGGCCTCCTGCACTGCTGATACCCGAAGCGACAGCCCCGATCCT534    GCTCCCACCCCGGCCCTGCCTATGCCTAAGGAGGATGCGCCTAGTGAC582    CCAGCACTGCCTGCTCCTCCACCAGCCACTGCTGTACACCTAAAACTG630    GTGCAGCCCTTTGTACGCAGAAGAAGTGCCCGCAGCCTGCAACACCTG678    TGCCGCCTTGTCATCAACCGTCTGGTGGCCGACGTGGACTGCCTGCCA726    CTGCCCCGGCGCATGGCCGACTACCTCCGACAGTACCCCTTCCAGCTC774    (2) INFORMATION FOR SEQ ID NO: 34:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 2249 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:    CGCGGCGTAGGACCTCCAACCCTACGAGAACAGGTTTTAGTTGAGCGAACGG52    GTGGACGCGCGGGCGCGGACGTTGCTGGACGTGCGGTGGTTCGACCGC100    GGCCCGTTCCTCTTCCTCGGGGACCTCAGCGTCATGGTCCACCCGGGC148    GATGACCCGTCGCCGCCGAAGCCGAGCCAGATGAGTCCGTAGGCGCAG196    AGGCTGTTGAACGGCCACCGGTAGTTTGTGCACCTCTTCCTGGCCTAA244    AGGCTGACCCCTCTCGACGGATTACCGTGAGCTCACGGGTACCTTCAC292    CAGGACGACTTCTTCCACTCGAGCCCAAAGAGGCCGCAGTAATCCGAG340    GGGCTCGGCCACGTTCTAGAGAAGCTGAAGTAGTGCCTTTCCCCTCGG388    GACGTTCTCCTCGACCGGGCGTCGAAGAAGACCGTCCACGACCTCCGG436    CACGCCGTGACGGTGTTGACGCCCCACGAGGTGGCGCTGTAGTTCCTG484    CTTTTGTAGGAATAGCTGGAGTTAGCGCCGCTCGAGTTCGAGTAGCTG532    AAGCCCAGCCCCCGCGACGAGTTCCTGTGGCAGATGTGCCTGAAGCTA580    CCCTGGGCTCACATATCGGGAGGTCTCACCTAGGCGATGGTAGCGATG628    GTACCGTCCAGCCGCCGTCAGACCAGGGACCCCTAGGACGACATACTA676    TACCACACACCTCTATAAGGAAAGCTCGTACTGCTTCTCTAGTAGTCC724    CCGGTCCAAAAGAAGTCCGTCTCCCAGAGAAGTCTTACAGTCGTAGAG772    TAATCTACCACGAACCGGGACTCTGGTAGTCTATCCGGTTGGAAGCTT820    CTTTAGGTCTTGGTAGGTACCTACGTTCTACAAGAGGACGGGGTCCTT868    TGACGACTCTAGGTGGAGGTGTCGGACAGCGGCCCCGGGTCGTTT913    ATCGTCGGAAAGACCGTCCAGGAGGGGAGAGAACAGTCTACGGGTCCTCCCTTCGAAGA972    CAGAGGTCGAAAGGGCTCATGGTCACTGTGCAGAGCGGTTCGTCCTGTCACGAACTATGT1032    CCTTGTTGTAAATGTTGAGTAAGGTCTAGGGTCCGGGGACCTCCGACGGAGGGTTGTCAC1092    CCCTTCTCACTGAGAGGTCCCCAGGATCCGGAGTTGAGGAGGGTATCTATGAGAGAAGAA1152    GAGTATCCACAGGTCGTAACGACCTGAGACTTTATAGGGCCCCCACCCCCCACCCCCACC1212    CAGTCTTGGGACGGTACCTTGACAAAGGAAGTAGTACTCAAGACGACTTACGGCGCTACC1272    CAGTCCATCCCCCCTTTGTCCAACCCTACCCTATCCTGATCGTGGTAAAATTCAGGGACA1332    GTGGAGAAGGCTGAGAAAGACTCACGGAAGACACCCCTGAGGCCGACACGACCCTCTTTA1392    TGAACTTGAACGGAGAAAATGGACGACGAAGAGGTTTTTAGACGGACCCAAAACAAGGGA1452    TAAAAAGAGAGGACAGGAGGGAGTGGGGGAGGAAGTATACTTTCCACGGTACCTTCTCCG1512    ATGTCCCGGTTTGCGACTCGGTGGACGGGAAAAAAGAGGAGGAAATCATTTTGAGGCTCA1572    CTTGACCAGAAGGAAAAACCAAAAATGAATTGACAAAGTTTCGGTTCTGGAGTGTGTGTG1632    TTTTTTACGTGTTTGTTACGTTAGTTGTCTTTTCGACATTTACACACATGTCAACCGTAC1692    CATCATATGTTTTTCTAACATCACCTAGATTAAAAATTCTTTAAAACGGAAATTCAATAA1752    AATGGACAAAAACAAAGAACAAAACTTTCTACGCGTAAGATTGGACCTCCAGTTACAATA1812    CATAAATAAATAAATAAATAAACCAAGGGAAGGATAAGGTTCGAAGCGACGACGGGATCA1872    AAAGAAAGGAGGAAAGGAGGAGACTGAACCCCTGGAAAACCCCCTCCCGACGCTGCGAAC1932    GAGACAAACACCCCACTGCCCTGAGTCCGCCCTGTCACGACGTCGAGGGACCGAAGACAC1992    CCCGGGGAGTGGATGAATGGGTCCACCCAGGGCCGAGACACCCACTACCCCTCCCCGTAA2052    CGACTGACACATATATCCTATTAATACTTTTCGTCAAGACCTACCACACGGAAGGTCTAG2112    GAGAGACCCCGACACAAAACTCGTCGTCCATCGGACGACCAAAATAGACTCACTTTATGA2172    CATGTCCCCTTATTTTCTCTAGAATAAAAAAAAAAATATGAACCGCAAAAAACTTATTTT2232    TGGAAAACAGAATTTTG2249    (2) INFORMATION FOR SEQ ID NO: 35:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 939 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:    ATGCTCTTGTCCAAAATCAACTCGCTTG28    CCCACCTGCGCGCCCGCGCCTGCAACGACCTGCACGCCACCAAGCTGGCG78    CCGGGCAAGGAGAAGGAGCCCCTGGAGTCGCAGTACCAGGTGGGCCCGCT128    ACTGGGCAGCGGCGGCTTCGGCTCGGTCTACTCAGGCATCCGCGTCTCCG178    ACAACTTGCCGGTGGCCATCAAACACGTGGAGAAGGACCGGATTTCCGAC228    TGGGGAGAGCTGCCTAATGGCACTCGAGTGCCCATGGAAGTGGTCCTGCT278    GAAGAAGGTGAGCTCGGGTTTCTCCGGCGTCATTAGGCTCCTGGACTGGT328    TCGAGAGGCCCGACAGTTTCGTCCTGATCCTGGAGAGGCCCGAGCCGGTG378    CAAGATCTCTTCGACTTCATCACGGAAAGGGGAGCCCTGCAAGAGGAGCT428    GGCCCGCAGCTTCTTCTGGCAGGTGCTGGAGGCCGTGCGGCACTGCCACA478    ACTGCGGGGTGCTCCACCGCGACATCAAGGACGAAAACATCCTTATCGAC528    CTCAATCGCGGCGAGCTCAAGCTCATCGACTTCGGGTCGGGGGCGCTGCT578    CAAGGACACCGTCTACACGGACTTCGATGGGACCCGAGTGTATAGCCCTC628    CAGAGTGGATCCGCTACCATCGCTACCATGGCAGGTCGGCGGCAGTCTGG678    TCCCTGGGGATCCTGCTGTATGATATGGTGTGTGGAGATATTCCTTTCGA728    GCATGACGAAGAGATCATCAGGGGCCAGGTTTTCTTCAGGCAGAGGGTCT778    CTTCAGAATGTCAGCATCTCATTAGATGGTGCTTGGCCCTGAGACCATCA828    GATAGGCCAACCTTCGAAGAAATCCAGAACCATCCATGGATGCAAGATGT878    TCTCCTGCCCCAGGAAACTGCTGAGATCCACCTCCACAGCCTGTCGCCGG928    GGCCCAGCAAA939    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We claim:
 1. A substantially pure polynucleotide, comprising a nucleicacid selected from the group consisting of(A) a sense nucleotide segmentencoding amino acids 1-60 of SEQ ID No: 4, alleles thereof, antibodybinding fragments thereof at least 10 amino acids long and fusionproteins thereof; (B) nucleotide segments which are anti-sense tosegments in (A); and (C) primers/probes at least 20 consecutivenucleotides long complementary to the segments of (A) and (B).
 2. Thepolynucleotide of claim 1, wherein the primers/probes comprise at least20 consecutive nucleotides of the sense nucleotide segment.
 3. Thepolynucleotide of claim 1, wherein the sense nucleotide segment encodesa fusion protein comprising (A) and an additional polynucleotide.
 4. Thepolynucleotide of claim 1, comprising a nucleic acid selected from thegroup consisting of a sense nucleotide segment and alleles thereofencoding a polypeptide selected from the group consisting of amino acids1-60 of SEQ. ID No: 4, its antibody binding, fragments thereof at least10 amino acids long, and fusion proteins thereof.
 5. The polynucleotideof claim 1, wherein the nucleic acid comprises an anti-sense nucleotidesegment.
 6. The polynucleotide of claim 1, wherein the nucleic acidcomprises an allele, or encodes and antibody binding fragment.
 7. Thepolynucleotide of claim 1, wherein the nucleotide segment encodes anantibody binding fragment at least 20 amino acids long.
 8. Thepolynucleotide of claim 1, comprising(A) primer/probes of at least 20consecutive nucleotides of the nucleic acid segment encoding apolypeptide selected from the group consisting of amino acids 1-60 ofSEQ. ID No: 4, and antibody binding fragments thereof; (B) allelesencoding amino acids 1-60 of SEQ. ID No: 4 or antibody binding fragmentsthereof at least 10 amino acids long; or (C) antisense polynucleotidesthereof.
 9. The polynucleotide of claim 1, wherein the nucleotidesegment comprises at least 20 consecutive nucleotides of SEQ. ID No: 3.10. The polynucleotide of claim 1, wherein the nucleic acid is selectedfrom the group consisting of(A) nucleotide segment selected from thegroup consisting of SEQ. ID No: 3, SEQ. ID No: 28, and alleles thereof;(B) nucleic acids encoding fusion proteins comprising nucleotidesegments and alleles of the segments encoding amino acids 1-60 of SEQ.ID No: 4; (C) primers/probes at least 20 consecutive nucleotides longcomplementary to the segments in (A) and (B); and (D) nucleotidesegments which are anti-sense to the nucleotide segments in (A), (B) or(C).
 11. The polynucleotide of claim 10, wherein the nucleic acidcomprises SEQ. ID No:
 1. 12. The polynucleotide of claim 10, wherein thenucleic acid comprises SEQ. ID No:
 28. 13. The polynucleotide of claim1, wherein the nucleic acid encodes amino acids 1-60 of SEQ. ID No: 4.14. The polynucleotide of claim 3, wherein the nucleic acid comprises anadditional polynucleotide encoding a polypeptide substantially unrelatedto amino acids 1-258 of SEQ. ID No:
 10. 15. The polynucleotide of claim13, wherein the additional polynucleotide comprises a nucleic acidselected from the group consisting of(A) sense nucleotide segments andalleles thereof encoding amino acids 1-202 of SEQ. ID No: 2, 1-358 ofSEQ. ID No: 6, 1-763 of SEQ. ID No: 8, 1-258 of SEQ ID NO: 10, 1-159 ofSEQ. ID No: 12, 1-412 of SEQ. ID No: 14, 1-313 of SEQ. ID No: 26,polymerase activating polypeptide, c-raf, c-fos, c-myc, c-myb, andpim-1, and antibody binding fragments thereof at least 10 amino acidslong; and (B) probes/primers at least 12 consecutive nucleotides longcomplementary to nucleotide segments in (A).
 16. The polynucleotide ofclaim 15, wherein the additional polynucleotide comprises a nucleic acidsegment selected from the group consisting of sense and anti-sensesegments and alleles thereof of SEQ. ID No: 1, SEQ. ID No: 5, SEQ. IDNo: 7, SEQ. ID No: 9, SEQ. ID No: 11, SEQ. ID No: 13, SEQ. ID No: 25,nucleic acid segments encoding polymerase activating polypeptide, c-raf,c-fos, c-myc, c-myb, and pim-1; probes/primers at least 12 consecutivenucleotides long complementary to the sense and anti-sense segments andalleles thereof; and fragments thereof at least 20 nucleotides long. 17.The polynucleotide of claim 1, being a DNA.
 18. The polynucleotide ofclaim 1, being an RNA.
 19. The polynucleotide of claim 1, wherein thenucleic acid comprises a probe/primer, and the probe/primer comprises anucleotide sequence at least 20 consecutive nucleotides longcomplementary to the sense or anti-sense nucleotide segments.
 20. Acomposition, comprising the polynucleotide of claim 1, and a diluent orcarrier.
 21. The composition of claim 20, where the diluent or carriercomprises a pharmaceutically acceptable diluent or carrier.
 22. Avector, comprising the polynucleotide of claim 1, linked in readingframe thereto.
 23. A composition, comprising the vector of claim 22, anda carrier or diluent.
 24. A vector comprising the polyribonucleotide ofclaim 4, linked in reading frame thereto.
 25. The vector of claim 24,comprising an expression vector.
 26. A host cell transfected with thevector of claim
 22. 27. A host cell transfected with the vector of claim25.
 28. A cell culture, comprising the hose cell of claim
 26. 29. A cellculture, comprising the host cell of claim
 27. 30. A method forproducing a polypeptide, comprising culturing a host cell of claim 27,in an expression medium, under conditions effective to express thepolypeptide; and separating the polypeptide from the cells.
 31. Themethod of claim 30, wherein the polypeptide is further separated fromthe medium.
 32. A method for producing a polypeptide, comprisingculturing a host cell of claim 26, in an expression medium, underconditions effective to express the polypeptide; and separating thepolypeptide from the cells.
 33. The method of claim 32, wherein thepolypeptide is further separated from the medium.
 34. A DNA, having anucleotide sequence complementary to the polynucleotide of claim
 1. 35.A RNA, having a polynucleotide sequence corresponding to the DNA ofclaim
 34. 36. A composition, comprising the DNA of claim 34, and acarrier or diluent.
 37. A composition, comprising the RNA of claim 35,and a carrier or diluent.
 38. A vector, comprising the DNA of claim 34linked thereto.
 39. A host cell, transfected with the vector of claim38.
 40. A cell culture, comprising a host cell comprising a vectorcomprising the probe/primer of claim
 19. 41. A composition, comprisingthe probe/primer of claim 19, and a carrier or diluent.
 42. A vector,comprising the probe/primer of claim 19 linked thereto.
 43. A host cell,transfected with the vector of claim
 42. 44. A hybridization kit,comprising the polynucleotide of claim 5, and instructions for its use.